Bi-Specific Monovalent Diabodies That are Capable of Binding CD123 and CD3, and Uses Thereof

ABSTRACT

The present invention is directed to sequence-optimized CD123×CD3 bi-specific monovalent diabodies that are capable of simultaneous binding to CD123 and CD3, and to the uses of such diabodies in the treatment of hematologic malignancies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/730,868 (filed Oct. 12, 2017), which is a continuation of U.S. patentapplication Ser. No. 14/913,632 (filed Feb. 22, 2016), which is a § 371National Stage Application of PCT/US2014/051790 (filed Aug. 20, 2014),which application claims priority to U.S. Patent Applications No.61/869,510 (filed Aug. 23, 2013), 61/907,749 (filed Nov. 22, 2013), and61/990,475 (filed May 8, 2014), and to European Patent Application No.13198784 (filed Dec. 20, 2013), each of which applications is hereinincorporated by reference in its entirety and to which priority isclaimed.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., submitted herewith as an ASCII text file SequenceListing (file name: 1301_0109PCT_SequenceListing.txt; created Aug. 21,2020; size 71,592 bytes) and incorporated herein by reference itsentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to CD123×CD3 bi-specific monovalentdiabodies that are capable of simultaneous binding to CD123 and CD3, andto the uses of such molecules in the treatment of hematologicmalignancies.

Description of Related Art I. CD123

CD123 (interleukin 3 receptor alpha, IL-3Ra) is a 40 kDa molecule and ispart of the interleukin 3 receptor complex (Stomski, F. C. et al. (1996)“Human Interleukin-3 (IL-3) Induces Disulfide-Linked IL-3 ReceptorAlpha-And Beta-Chain Heterodimerization, Which Is Required For ReceptorActivation But Not High Affinity Binding,” Mol. Cell. Biol.16(6):3035-3046). Interleukin 3 (IL-3) drives early differentiation ofmultipotent stem cells into cells of the erythroid, myeloid and lymphoidprogenitors. CD123 is expressed on CD34+ committed progenitors (Taussig,D. C. et al. (2005) “Hematopoietic Stem Cells Express Multiple MyeloidMarkers: Implications For The Origin And Targeted Therapy Of AcuteMyeloid Leukemia,” Blood 106:4086-4092), but not by CD34+/CD38− normalhematopoietic stem cells. CD123 is expressed by basophils, mast cells,plasmacytoid dendritic cells, some expression by monocytes, macrophagesand eosinophils, and low or no expression by neutrophils andmegakaryocytes. Some non-hematopoietic tissues (placenta, Leydig cellsof the testis, certain brain cell elements and some endothelial cells)express CD123; however expression is mostly cytoplasmic.

CD123 is reported to be expressed by leukemic blasts and leukemia stemcells (LSC) (Jordan, C. T. et al. (2000) “The Interleukin-3 ReceptorAlpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia StemCells,” Leukemia 14:1777-1784; Jin, W. et al. (2009) “Regulation Of Th17Cell Differentiation And EAE Induction By MAP3K NIK,” Blood113:6603-6610) (FIG. 1). In human normal precursor populations, CD123 isexpressed by a subset of hematopoietic progenitor cells (HPC) but not bynormal hematopoietic stem cells (HSC). CD123 is also expressed byplasmacytoid dendritic cells (pDC) and basophils, and, to a lesserextent, monocytes and eosinophils (Lopez, A. F. et al. (1989)“Reciprocal Inhibition Of Binding Between Interleukin 3 And GranulocyteMacrophage Colony-Stimulating Factor To Human Eosinophils,” Proc. Natl.Acad. Sci. (U.S.A.) 86:7022-7026; Sun, Q. et al. (1996) “MonoclonalAntibody 7G3 Recognizes The N-Terminal Domain Of The Human Interleukin-3(IL-3) Receptor Alpha Chain And Functions As A Specific IL-3 ReceptorAntagonist,” Blood 87:83-92; Muñoz, L. et al. (2001) “Interleukin-3Receptor Alpha Chain (CD123) Is Widely Expressed In HematologicMalignancies,” Haematologica 86(12):1261-1269; Masten, B. J. et al.(2006) “Characterization Of Myeloid And Plasmacytoid Dendritic Cells InHuman Lung,” J. Immunol. 177:7784-7793; Korpelainen, E. I. et al. (1995)“Interferon-Gamma Upregulates Interleukin-3 (IL-3) Receptor ExpressionIn Human Endothelial Cells And Synergizes With IL-3 In Stimulating MajorHistocompatibility Complex Class II Expression And Cytokine Production,”Blood 86:176-182).

CD123 has been reported to be overexpressed on malignant cells in a widerange of hematologic malignancies including acute myeloid leukemia (AML)and myelodysplastic syndrome (MDS) (Munoz, L. et al. (2001)“Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed InHematologic Malignancies,” Haematologica 86(12):1261-1269).Overexpression of CD123 is associated with poorer prognosis in AML(Tettamanti, M. S. et al. (2013) “Targeting Of Acute Myeloid LeukaemiaBy Cytokine-Induced Killer Cells Redirected With A Novel CD123-SpecificChimeric Antigen Receptor,” Br. J. Haematol. 161:389-401).

AML and MDS are thought to arise in and be perpetuated by a smallpopulation of leukemic stem cells (LSCs), which are generally dormant(i.e., not rapidly dividing cells) and therefore resist cell death(apoptosis) and conventional chemotherapeutic agents. LSCs arecharacterized by high levels of CD123 expression, which is not presentin the corresponding normal hematopoietic stem cell population in normalhuman bone marrow (Jin, W. et al. (2009) “Regulation Of Th17 CellDifferentiation And EAE Induction By MAP3K NIK,” Blood 113:6603-6610;Jordan, C. T. et al. (2000) “The Interleukin-3 Receptor Alpha Chain Is AUnique Marker For Human Acute Myelogenous Leukemia Stem Cells,” Leukemia14:1777-1784). CD123 is expressed in 45%-95% of AML, 85% of Hairy cellleukemia (HCL), and 40% of acute B lymphoblastic leukemia (B-ALL). CD123expression is also associated with multiple othermalignancies/pre-malignancies: chronic myeloid leukemia (CML) progenitorcells (including blast crisis CML); Hodgkin's Reed Sternberg (RS) cells;transformed non-Hodgkin's lymphoma (NHL); some chronic lymphocyticleukemia (CLL) (CD11c+); a subset of acute T lymphoblastic leukemia(T-ALL) (16%, most immature, mostly adult), plasmacytoid dendritic cell(pDC) (DC2) malignancies and CD34+/CD38− myelodysplastic syndrome (MDS)marrow cell malignancies.

AML is a clonal disease characterized by the proliferation andaccumulation of transformed myeloid progenitor cells in the bone marrow,which ultimately leads to hematopoietic failure. The incidence of AMLincreases with age, and older patients typically have worse treatmentoutcomes than do younger patients (Robak, T. et al. (2009) “Current AndEmerging Therapies For Acute Myeloid Leukemia,” Clin. Ther.2:2349-2370). Unfortunately, at present, most adults with AML die fromtheir disease.

Treatment for AML initially focuses in the induction of remission(induction therapy). Once remission is achieved, treatment shifts tofocus on securing such remission (post-remission or consolidationtherapy) and, in some instances, maintenance therapy. The standardremission induction paradigm for AML is chemotherapy with ananthracycline/cytarabine combination, followed by either consolidationchemotherapy (usually with higher doses of the same drugs as were usedduring the induction period) or human stem cell transplantation,depending on the patient's ability to tolerate intensive treatment andthe likelihood of cure with chemotherapy alone (see, e.g., Roboz, G. J.(2012) “Current Treatment Of Acute Myeloid Leukemia,” Curr. Opin. Oncol.24:711-719).

Agents frequently used in induction therapy include cytarabine and theanthracyclines. Cytarabine, also known as AraC, kills cancer cells (andother rapidly dividing normal cells) by interfering with DNA synthesis.Side effects associated with AraC treatment include decreased resistanceto infection, a result of decreased white blood cell production;bleeding, as a result of decreased platelet production; and anemia, dueto a potential reduction in red blood cells. Other side effects includenausea and vomiting. Anthracyclines (e.g., daunorubicin, doxorubicin,and idarubicin) have several modes of action including inhibition of DNAand RNA synthesis, disruption of higher order structures of DNA, andproduction of cell damaging free oxygen radicals. The most consequentialadverse effect of anthracyclines is cardiotoxicity, which considerablylimits administered life-time dose and to some extent their usefulness.

Thus, unfortunately, despite substantial progress in the treatment ofnewly diagnosed AML, 20% to 40% of patients do not achieve remissionwith the standard induction chemotherapy, and 50% to 70% of patientsentering a first complete remission are expected to relapse within 3years. The optimum strategy at the time of relapse, or for patients withthe resistant disease, remains uncertain. Stem cell transplantation hasbeen established as the most effective form of anti-leukemic therapy inpatients with AML in first or subsequent remission (Roboz, G. J. (2012)“Current Treatment Of Acute Myeloid Leukemia,” Curr. Opin. Oncol.24:711-719).

II. CD3

CD3 is a T cell co-receptor composed of four distinct chains(Wucherpfennig, K. W. et al. (2010) “Structural Biology Of The T-CellReceptor: Insights Into Receptor Assembly, Ligand Recognition, AndInitiation Of Signaling,” Cold Spring Harb. Perspect. Biol.2(4):a005140; pages 1-14). In mammals, the complex contains a CD3γchain, a CD3δ chain, and two CD3ε chains. These chains associate with amolecule known as the T cell receptor (TCR) in order to generate anactivation signal in T lymphocytes. In the absence of CD3, TCRs do notassemble properly and are degraded (Thomas, S. et al. (2010) “MolecularImmunology Lessons From Therapeutic T-Cell Receptor Gene Transfer,”Immunology 129(2):170-177). CD3 is found bound to the membranes of allmature T cells, and in virtually no other cell type (see, Janeway, C. A.et al. (2005) In: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH ANDDISEASE,” 6th ed. Garland Science Publishing, NY, pp. 214-216; Sun, Z.J. et al. (2001) “Mechanisms Contributing To T Cell Receptor SignalingAnd Assembly Revealed By The Solution Structure Of An EctodomainFragment Of The CD3ε:γ Heterodimer,” Cell 105(7):913-923; Kuhns, M. S.et al. (2006) “Deconstructing The Form And Function Of The TCR/CD3Complex,” Immunity. 2006 February; 24(2):133-139).

III. Bi-Specific Diabodies

The ability of an intact, unmodified antibody (e.g., an IgG) to bind anepitope of an antigen depends upon the presence of variable domains onthe immunoglobulin light and heavy chains (i.e., the VL and VH domains,respectively). The design of a diabody is based on the single chain Fvconstruct (scFv) (see, e.g., Holliger et al. (1993) “‘Diabodies’: SmallBivalent And Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci.(U.S.A.) 90:6444-6448; US 2004/0058400 (Holliger et al.); US2004/0220388 (Mertens et al.); Alt et al. (1999) FEBS Lett.454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-LikeBispecific Antibody To Both The Epidermal Growth Factor Receptor And TheInsulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J.Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); Olafsen,T. et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody AllowsSite-Specific Conjugation And Radiolabeling For Tumor TargetingApplications,” Protein Eng. Des. Sel. 17(1):21-27; Wu, A. et al. (2001)“Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fc FusionProtein Is Mediated Through Variable Domain Exchange,” ProteinEngineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For CancerImmunotherapy And Its Functional Enhancement By Fusion Of Human FcDomain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al.(2000) “Construction Of A Diabody (Small Recombinant BispecificAntibody) Using A Refolding System,” Protein Eng. 13(8):583-588;Baeuerle, P. A. et al. (2009) “Bispecific T-Cell Engaging Antibodies ForCancer Therapy,” Cancer Res. 69(12):4941-4944).

Interaction of an antibody light chain and an antibody heavy chain and,in particular, interaction of its VL and VH domains forms one of theepitope binding sites of the antibody. In contrast, the scFv constructcomprises a VL and VH domain of an antibody contained in a singlepolypeptide chain wherein the domains are separated by a flexible linkerof sufficient length to allow self-assembly of the two domains into afunctional epitope binding site. Where self-assembly of the VL and VHdomains is rendered impossible due to a linker of insufficient length(less than about 12 amino acid residues), two of the scFv constructsinteract with one another other to form a bivalent molecule in which theVL of one chain associates with the VH of the other (reviewed in Marvinet al. (2005) “Recombinant Approaches To IgG-Like BispecificAntibodies,” Acta Pharmacol. Sin. 26:649-658).

Natural antibodies are capable of binding to only one epitope species(i.e., mono-specific), although they can bind multiple copies of thatspecies (i.e., exhibiting bi-valency or multi-valency). The art hasnoted the capability to produce diabodies that differ from such naturalantibodies in being capable of binding two or more different epitopespecies (i.e., exhibiting bi-specificity or multispecificity in additionto bi-valency or multi-valency) (see, e.g., Holliger et al. (1993)“‘Diabodies’: Small Bivalent And Bispecific Antibody Fragments,” Proc.Natl. Acad. Sci. (U.S.A.) 90:6444-6448; US 2004/0058400 (Holliger etal.); US 2004/0220388 (Mertens et al.); Alt et al. (1999) FEBS Lett.454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-LikeBispecific Antibody To Both The Epidermal Growth Factor Receptor And TheInsulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J.Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); Mertens,N. et al., “New Recombinant Bi- and Trispecific Antibody Derivatives,”In: NOVEL FRONTIERS IN THE PRODUCTION OF COMPOUNDS FOR BIOMEDICAL USE,A. VanBroekhoven et al. (Eds.), Kluwer Academic Publishers, Dordrecht,The Netherlands (2001), pages 195-208; Wu, A. et al. (2001)“Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fc FusionProtein Is Mediated Through Variable Domain Exchange,” ProteinEngineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For CancerImmunotherapy And Its Functional Enhancement By Fusion Of Human FcDomain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al.(2000) “Construction Of A Diabody (Small Recombinant BispecificAntibody) Using A Refolding System,” Protein Eng. 13(8):583-588;Baeuerle, P. A. et al. (2009) “Bispecific T-Cell Engaging Antibodies ForCancer Therapy,” Cancer Res. 69(12):4941-4944).

The provision of non-monospecific diabodies provides a significantadvantage: the capacity to co-ligate and co-localize cells that expressdifferent epitopes. Bi-specific diabodies thus have wide-rangingapplications including therapy and immunodiagnosis. Bi-specificityallows for great flexibility in the design and engineering of thediabody in various applications, providing enhanced avidity tomultimeric antigens, the cross-linking of differing antigens, anddirected targeting to specific cell types relying on the presence ofboth target antigens. Due to their increased valency, low dissociationrates and rapid clearance from the circulation (for diabodies of smallsize, at or below ˜50 kDa), diabody molecules known in the art have alsoshown particular use in the field of tumor imaging (Fitzgerald et al.(1997) “Improved Tumour Targeting By Disulphide Stabilized DiabodiesExpressed In Pichia pastoris,” Protein Eng. 10:1221). Of particularimportance is the co-ligating of differing cells, for example, thecross-linking of cytotoxic T cells to tumor cells (Staerz et al. (1985)“Hybrid Antibodies Can Target Sites For Attack By T Cells,” Nature314:628-631, and Holliger et al. (1996) “Specific Killing Of LymphomaCells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” ProteinEng. 9:299-305).

Diabody epitope binding domains may also be directed to a surfacedeterminant of any immune effector cell such as CD3, CD16, CD32, orCD64, which are expressed on T lymphocytes, natural killer (NK) cells orother mononuclear cells. In many studies, diabody binding to effectorcell determinants, e.g., Fcγ receptors (FcγR), was also found toactivate the effector cell (Holliger et al. (1996) “Specific Killing OfLymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,”Protein Eng. 9:299-305; Holliger et al. (1999) “Carcinoembryonic Antigen(CEA)-Specific T-cell Activation In Colon Carcinoma Induced ByAnti-CD3×Anti-CEA Bispecific Diabodies And B7×Anti-CEA Bispecific FusionProteins,” Cancer Res. 59:2909-2916; WO 2006/113665; WO 2008/157379; WO2010/080538; WO 2012/018687; WO 2012/162068). Normally, effector cellactivation is triggered by the binding of an antigen bound antibody toan effector cell via Fc-FcγR interaction; thus, in this regard, diabodymolecules may exhibit Ig-like functionality independent of whether theycomprise an Fc Domain (e.g., as assayed in any effector function assayknown in the art or exemplified herein (e.g., ADCC assay)). Bycross-linking tumor and effector cells, the diabody not only brings theeffector cell within the proximity of the tumor cells but leads toeffective tumor killing (see e.g., Cao et al. (2003) “BispecificAntibody Conjugates In Therapeutics,” Adv. Drug. Deliv. Rev.55:171-197).

However, the above advantages come at a salient cost. The formation ofsuch non-monospecific diabodies requires the successful assembly of twoor more distinct and different polypeptides (i.e., such formationrequires that the diabodies be formed through the heterodimerization ofdifferent polypeptide chain species). This fact is in contrast tomono-specific diabodies, which are formed through the homodimerizationof identical polypeptide chains. Because at least two dissimilarpolypeptides (i.e., two polypeptide species) must be provided in orderto form a non-monospecific diabody, and because homodimerization of suchpolypeptides leads to inactive molecules (Takemura, S. et al. (2000)“Construction Of A Diabody (Small Recombinant Bispecific Antibody) UsingA Refolding System,” Protein Eng. 13(8):583-588), the production of suchpolypeptides must be accomplished in such a way as to prevent covalentbonding between polypeptides of the same species (i.e., so as to preventhomodimerization) (Takemura, S. et al. (2000) “Construction Of A Diabody(Small Recombinant Bispecific Antibody) Using A Refolding System,”Protein Eng. 13(8):583-588). The art has therefore taught thenon-covalent association of such polypeptides (see, e.g., Olafsen et al.(2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-SpecificConjugation And Radiolabeling For Tumor Targeting Applications,” Prot.Engr. Des. Sel. 17:21-27; Asano et al. (2004) “A Diabody For CancerImmunotherapy And Its Functional Enhancement By Fusion Of Human FcDomain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al.(2000) “Construction Of A Diabody (Small Recombinant BispecificAntibody) Using A Refolding System,” Protein Eng. 13(8):583-588; Lu, D.et al. (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody ToBoth The Epidermal Growth Factor Receptor And The Insulin-Like GrowthFactor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem.280(20):19665-19672).

However, the art has recognized that bi-specific diabodies composed ofnon-covalently associated polypeptides are unstable and readilydissociate into non-functional monomers (see, e.g., Lu, D. et al. (2005)“A Fully Human Recombinant IgG-Like Bispecific Antibody To Both TheEpidermal Growth Factor Receptor And The Insulin-Like Growth FactorReceptor For Enhanced Antitumor Activity,” J. Biol. Chem.280(20):19665-19672).

In the face of this challenge, the art has succeeded in developingstable, covalently bonded heterodimeric non-monospecific diabodies (see,e.g., WO 2006/113665; WO/2008/157379; WO 2010/080538; WO 2012/018687;WO/2012/162068; Johnson, S. et al. (2010) “Effector Cell RecruitmentWith Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To PotentTumor Cytolysis And In Vivo B-Cell Depletion,” J. Molec. Biol.399(3):436-449; Veri, M. C. et al. (2010) “Therapeutic Control Of B CellActivation Via Recruitment Of Fcgamma Receptor IIb (CD32B) InhibitoryFunction With A Novel Bispecific Antibody Scaffold,” Arthritis Rheum.62(7):1933-1943; Moore, P. A. et al. (2011) “Application Of DualAffinity Retargeting Molecules To Achieve Optimal Redirected T-CellKilling Of B-Cell Lymphoma,” Blood 117(17):4542-4551). Such approachesinvolve engineering one or more cysteine residues into each of theemployed polypeptide species. For example, the addition of a cysteineresidue to the C-terminus of such constructs has been shown to allowdisulfide bonding between the polypeptide chains, stabilizing theresulting heterodimer without interfering with the bindingcharacteristics of the bivalent molecule.

Notwithstanding such success, the production of stable, functionalheterodimeric, non-monospecific diabodies can be further optimized bythe careful consideration and placement of cysteine residues in one ormore of the employed polypeptide chains. Such optimized diabodies can beproduced in higher yield and with greater activity than non-optimizeddiabodies. The present invention is thus directed to the problem ofproviding polypeptides that are particularly designed and optimized toform heterodimeric diabodies. The invention solves this problem throughthe provision of exemplary, optimized CD123×CD3 diabodies.

SUMMARY OF THE INVENTION

The present invention is directed to CD123×CD3 bi-specific diabodiesthat are capable of simultaneous binding to CD123 and CD3, and to theuses of such molecules in the treatment of disease, in particularhematologic malignancies.

The CD123×CD3 bi-specific diabodies of the invention comprise at leasttwo different polypeptide chains that associate with one another in aheterodimeric manner to form one binding site specific for an epitope ofCD123 and one binding site specific for an epitope of CD3. A CD123×CD3diabody of the invention is thus monovalent in that it is capable ofbinding to only one copy of an epitope of CD123 and to only one copy ofan epitope of CD3, but bi-specific in that a single diabody is able tobind simultaneously to the epitope of CD123 and to the epitope of CD3.The individual polypeptide chains of the diabodies are covalently bondedto one another, for example by disulfide bonding of cysteine residueslocated within each polypeptide chain. In particular embodiments, thediabodies of the present invention further have an immunoglobulin FcDomain or an Albumin-Binding Domain to extend half-life in vivo.

In detail, the invention also provides a sequence-optimized CD123×CD3bi-specific monovalent diabody capable of specific binding to an epitopeof CD123 and to an epitope of CD3, wherein the diabody comprises a firstpolypeptide chain and a second polypeptide chain, covalently bonded toone another, wherein:

-   A. the first polypeptide chain comprises, in the N-terminal to    C-terminal direction:    -   i. a Domain 1, comprising:        -   (1) a sub-Domain (1A), which comprises a VL Domain of a            monoclonal antibody capable of binding to CD3 (VL_(CD3))            (SEQ ID NO:21); and        -   (2) a sub-Domain (1B), which comprises a VH Domain of a            monoclonal antibody capable of binding to CD123 (VH_(CD123))            (SEQ ID NO:26);    -    wherein the sub-Domains 1A and 1B are separated from one        another by a peptide linker (SEQ ID NO:29);    -   ii. a Domain 2, wherein the Domain 2 is an E-coil Domain (SEQ ID        NO:34) or a K-coil Domain (SEQ ID NO:35), wherein the Domain 2        is separated from the Domain 1 by a peptide linker (SEQ ID        NO:30); and-   B. the second polypeptide chain comprises, in the N-terminal to    C-terminal direction:    -   i. a Domain 1, comprising:        -   (1) a sub-Domain (1A), which comprises a VL Domain of a            monoclonal antibody capable of binding to CD123 (VL_(CD123))            (SEQ ID NO:25); and        -   (2) a sub-Domain (1B), which comprises a VH Domain of a            monoclonal antibody capable of binding to CD3 (VH_(CD3))            (SEQ ID NO:22);    -    wherein the sub-Domains 1A and 1B are separated from one        another by a peptide linker (SEQ ID NO:29);    -   ii. a Domain 2, wherein the Domain 2 is a K-coil Domain (SEQ ID        NO:35) or an E-coil Domain (SEQ ID NO:34), wherein the Domain 2        is separated from the Domain 1 by a peptide linker (SEQ ID        NO:30); and wherein the Domain 2 of the first and the second        polypeptide chains are not both E-coil Domains or both K-coil        Domains;        and wherein:-   (a) said VL Domain of said first polypeptide chain and said VH    Domain of said second polypeptide chain form an Antigen Binding    Domain capable of specifically binding to an epitope of CD3; and-   (b) said VL Domain of said second polypeptide chain and said VH    Domain of said first polypeptide chain form an Antigen Binding    Domain capable of specifically binding to an epitope of CD123.

The invention also provides a non-sequence-optimized CD123×CD3bi-specific monovalent diabody capable of specific binding to an epitopeof CD123 and to an epitope of CD3, wherein the diabody comprises a firstpolypeptide chain and a second polypeptide chain, covalently bonded toone another, wherein:

-   A. the first polypeptide chain comprises, in the N-terminal to    C-terminal direction:    -   i. a Domain 1, comprising:        -   (1) a sub-Domain (1A), which comprises a VL Domain of a            monoclonal antibody capable of binding to CD3 (VL_(CD3))            (SEQ ID NO:23); and        -   (2) a sub-Domain (1B), which comprises a VH Domain of a            monoclonal antibody capable of binding to CD123 (VH_(CD123))            (SEQ ID NO:28);    -    wherein the sub-Domains 1A and 1B are separated from one        another by a peptide linker (SEQ ID NO:29);    -   ii. a Domain 2, wherein the Domain 2 is an E-coil Domain (SEQ ID        NO:34) or a K-coil Domain (SEQ ID NO:35), wherein the Domain 2        is separated from the Domain 1 by a peptide linker (SEQ ID        NO:30); and-   B. the second polypeptide chain comprises, in the N-terminal to    C-terminal direction:    -   i. a Domain 1, comprising:        -   (1) a sub-Domain (1A), which comprises a VL Domain of a            monoclonal antibody capable of binding to CD123 (VL_(CD23))            (SEQ ID NO:27); and        -   (2) a sub-Domain (1B), which comprises a VH Domain of a            monoclonal antibody capable of binding to CD3 (VH_(CD3))            (SEQ ID NO:24);    -    wherein the sub-Domains 1A and 1B are separated from one        another by a peptide linker (SEQ ID NO:29);    -   ii. a Domain 2, wherein the Domain 2 is a K-coil Domain (SEQ ID        NO:35) or an E-coil Domain (SEQ ID NO:34), wherein the Domain 2        is separated from the Domain 1 by a peptide linker (SEQ ID        NO:30); and wherein the Domain 2 of the first and the second        polypeptide chains are not both E-coil Domains or both K-coil        Domains        and wherein:-   (a) said VL Domain of said first polypeptide chain and said VH    Domain of said second polypeptide chain form an Antigen Binding    Domain capable of specifically binding to an epitope of CD3; and-   (b) said VL Domain of said second polypeptide chain and said VH    Domain of said first polypeptide chain form an Antigen Binding    Domain capable of specifically binding to an epitope of CD123.

The invention additionally provides the embodiment of theabove-described bi-specific monovalent diabodies, wherein the first orsecond polypeptide chain additionally comprises an Albumin-BindingDomain (SEQ ID NO:36) linked, C-terminally to Domain 2 or N-terminallyto Domain 1, via a peptide linker (SEQ ID NO:31).

The invention additionally provides the embodiment of theabove-described bi-specific monovalent diabodies wherein the first orsecond polypeptide chain additionally comprises a Domain 3 comprising aCH2 and CH3 Domain of an immunoglobulin IgG Fc Domain (SEQ ID NO:37),wherein the Domain 3 is linked, N-terminally, to the Domain 1A via apeptide linker (SEQ ID NO:33).

The invention additionally provides the embodiment of theabove-described bi-specific monovalent diabodies wherein the first orsecond polypeptide chain additionally comprises a Domain 3 comprising aCH2 and CH3 Domain of an immunoglobulin IgG Fc Domain (SEQ ID NO:37),wherein the Domain 3 is linked, C-terminally, to the Domain 2 via apeptide linker (SEQ ID NO:32).

The invention additionally provides the embodiment of any of theabove-described bi-specific monovalent diabodies wherein the Domain 2 ofthe first polypeptide chain is a K-coil Domain (SEQ ID NO:35) and theDomain 2 of the second polypeptide chain is an E-coil Domain (SEQ IDNO:34).

The invention additionally provides the embodiment of any of theabove-described bi-specific monovalent diabodies wherein the Domain 2 ofthe first polypeptide chain is an E-coil Domain (SEQ ID NO:34) and theDomain 2 of the second polypeptide chain is a K-coil Domain (SEQ IDNO:35).

The invention additionally provides the embodiment of a bi-specificmonovalent diabody capable of specific binding to an epitope of CD123and to an epitope of CD3, wherein the diabody comprises a firstpolypeptide chain and a second polypeptide chain, covalently bonded toone another, wherein: said bi-specific diabody comprises:

-   -   A. a first polypeptide chain having the amino acid sequence of        SEQ ID NO:1; and    -   B. a second polypeptide chain having the amino acid sequence of        SEQ ID NO:3;        wherein said first and said second polypeptide chains are        covalently bonded to one another by a disulfide bond.

The diabodies of the invention exhibit unexpectedly enhanced functionalactivities as further described below.

The diabodies of the invention are preferably capable of cross-reactingwith both human and primate CD123 and CD3 proteins, preferablycynomolgus monkey CD123 and CD3 proteins.

The diabodies of the invention are preferably capable of depleting, inan in vitro cell-based assay, plasmacytoid dendritic cells (pDC) from aculture of primary PBMCs with an IC50 of about 1 ng/ml or less, about0.8 ng/ml or less, about 0.6 ng/ml or less, about 0.4 ng/ml or less,about 0.2 ng/ml or less, about 0.1 ng/ml or less, about 0.05 ng/ml orless, about 0.04 ng/ml or less, about 0.03 ng/ml or less, about 0.02ng/ml or less or about 0.01 ng/ml or less. Preferably, the IC50 is about0.01 ng/ml or less. In the above-described assay, the culture of primaryPBMCs may be from cynomolgus monkey in which case said depletion is ofcynomolgus monkey plasmacytoid dendritic cells (pDC). Optionally thediabodies of the invention may be capable of depleting plasmacytoiddendritic cells (pDC) from a primary culture of PBMCs as described abovewherein the assay is conducted by or in accordance with the protocol ofExample 14, as herein described, or by modification of such assay aswould be understood by those of ordinary skill, or by other means knownto those of ordinary skill.

The diabodies of the invention preferably exhibit cytotoxicity in an invitro Kasumi-3 assay with an EC50 of about 0.05 ng/mL or less.Preferably, the EC50 is about 0.04 ng/mL or less, about 0.03 ng/mL orless, about 0.02 ng/mL or less, or about 0.01 ng/mL or less. Optionallythe diabodies of the invention may exhibit cytotoxicity as describedabove wherein the assay is conducted by or in accordance with theprotocol of Example 3 as herein described, or by modification of suchassay as would be understood by those of ordinary skill, or by othermeans known to those of ordinary skill.

The diabodies of the invention preferably exhibit cytotoxicity in an invitro Molm-13 assay with an EC50 of about 5 ng/mL or less. Preferably,the EC50 is about 3 ng/mL or less, about 2 ng/mL or less, about 1 ng/mLor less, about 0.75 ng/mL or less, or about 0.2 ng/mL or less.Optionally the diabodies of the invention may exhibit cytotoxicity asdescribed above wherein the assay is conducted by or in accordance withthe protocol of Example 3 as herein described, or by modification ofsuch assay as would be understood by those of ordinary skill, or byother means known to those of ordinary skill.

The diabodies of the invention are preferably capable of inhibiting thegrowth of a MOLM-13 tumor xenograft in a mouse. Preferably the diabodiesof the invention may be capable of inhibiting the growth of a MOLM-13tumor xenograft in a mouse at a concentration of at least about 20μg/kg, at least about 4 μg/kg, at least about 0.8 μg/kg, at least about0.6 μg/kg or at least about 0.4 μg/kg. Preferred antibodies of theinvention will inhibit growth of a MOLM-13 tumor xenograft in a mouse byat least 25%, but possibly by at least about 40% or more, by at leastabout 50% or more, by at least about 60% or more, by at least about 70%or more, by at least about 80% or more, by at least about 90% or more,or even by completely inhibiting MOLM-13 tumor growth after some periodof time or by causing tumor regression or disappearance. This inhibitionwill take place for at least an NSG mouse strain. Optionally, thediabodies of the invention may be capable of inhibiting the growth of aMOLM-13 tumor xenograft in a mouse in the above-described manner by orin accordance with the protocol of Example 6 as herein described, or bymodification of such assay as would be understood by those of ordinaryskill, or by other means known to those of ordinary skill.

The diabodies of the invention are preferably capable of inhibiting thegrowth of an RS4-11 tumor xenograft in a mouse. Preferably the diabodiesof the invention may be capable of inhibiting the growth of a RS4-11tumor xenograft in a mouse at a concentration of at least about 0.5mg/kg, at least about 0.2 mg/kg, at least about 0.1 mg/kg, at leastabout 0.02 mg/kg or at least about 0.004 mg/kg. Preferred antibodies ofthe invention will inhibit growth of a RS4-11 tumor xenograft in a mouseby at least about 25%, but possibly at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, or even by completely inhibiting RS4-11 tumor growthafter some period of time or by causing tumor regression ordisappearance. This inhibition will take place for at least an NSG mousestrain. Optionally, the diabodies of the invention may be capable ofinhibiting the growth of a RS4-11 tumor xenograft in a mouse in theabove-described manner by or in accordance with the protocol of Example6 as herein described, or by modification of such assay as would beunderstood by those of ordinary skill, or by other means known to thoseof ordinary skill.

The diabodies of the invention are preferably capable of depletingleukemic blast cells in vitro in a primary culture of AML bone marrowcells. Preferably the diabodies of the invention may be capable ofdepleting leukemic blast cells in vitro in a primary culture of AML bonemarrow cells at concentrations of at least about 0.01 ng/ml, at leastabout 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml,at least about 0.08 ng/ml or at least about 0.1 ng/ml. Preferably, thediabodies of the invention may be capable of depleting leukemic blastcells in vitro in a primary culture of AML bone marrow cells to lessthan 20% of the total population of primary leukemic blast cells atdiabody concentrations of at least about 0.01 ng/ml, at least about 0.02ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at leastabout 0.08 ng/ml or at least about 0.1 ng/ml, optionally followingincubation of the primary culture with diabody for about 120 hours.Preferably leukemic blast cells are depleted in vitro in a primaryculture of AML bone marrow cells to less than 20% of the totalpopulation of primary leukemic blast cells at diabody concentrations ofabout 0.01 ng/ml or 0.1 ng/ml following incubation of the primaryculture with diabody for about 120 hours.

The diabodies of the invention are preferably capable of inducing anexpansion of a T cell population in vitro in a primary culture of AMLbone marrow cells. Preferably, such expansion may be to about 70% ormore of the maximum T cell population which can be expanded in theassay. Preferably the diabodies of the invention may be capable ofinducing an expansion of a T cell population in vitro in a primaryculture of AML bone marrow cells to about 70% or more of the maximum Tcell population which can be expanded in the assay at diabodyconcentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml,at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about0.08 ng/ml or at least about 0.1 ng/ml, optionally following incubationof the primary culture with diabody for about 120 hours. Preferably, a Tcell population is expanded in vitro in a primary culture of AML bonemarrow cells to about 70% or more of the maximum T cell population whichcan be expanded in the assay at diabody concentrations of about 0.01ng/ml or about 0.1 ng/ml following incubation of the primary culturewith diabody for about 120 hours.

The diabodies of the invention are preferably capable of inducing anactivation of a T cell population in vitro in a primary culture of AMLbone marrow cells. Such activation may occur at diabody concentrationsof at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or atleast about 0.1 ng/ml, optionally following incubation of the primaryculture with diabody for about 72 hours. Such activation may be measuredby the expression of a T cell activation marker such as CD25.Preferably, activation of a T cell population in vitro in a primaryculture of AML bone marrow cells as measured by expression of CD25 mayoccur at diabody concentrations of about 0.01 ng/ml or about 0.1 ng/mlfollowing incubation of the primary culture with diabody for about 72hours.

The diabodies of the invention are preferably capable of depletingleukemic blast cells in vitro in a primary culture of AML bone marrowcells to less than 20% of the total population of primary leukemic blastcells and at the same time inducing an expansion of the T cellpopulation in vitro in the primary culture of AML bone marrow cells toabout 70% or more of the maximum T cell population which can be expandedin the assay at diabody concentrations of at least about 0.01 ng/ml, atleast about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06ng/ml, at least about 0.08 ng/ml or at least about 0.1 ng/ml, optionallyfollowing incubation of the primary culture with diabody for about 120hours. Preferably, the diabody concentrations are about 0.01 ng/ml orabout 0.1 ng/ml and the primary culture is incubated with diabody forabout 120 hours.

The diabodies of the invention may be capable of depleting leukemicblast cells in vitro in a primary culture of AML bone marrow cellsand/or inducing an expansion of a T cell population in vitro in aprimary culture of AML bone marrow cells and/or inducing an activationof a T cell population in vitro in a primary culture of AML bone marrowcells in the above-described manner by or in accordance with theprotocol of Example 8 as herein described, or by modification of suchassay as would be understood by those of ordinary skill, or by othermeans known to those of ordinary skill.

For the avoidance of any doubt, the diabodies of the invention mayexhibit one, two, three, more than three or all of the functionalattributes described herein. Thus the diabodies of the invention mayexhibit any combination of the functional attributes described herein.

The diabodies of the invention may be for use as a pharmaceutical.

Preferably, the diabodies are for use in the treatment of a disease orcondition associated with or characterized by the expression of CD123.The invention also relates to the use of diabodies of the invention inthe manufacture of a pharmaceutical composition, preferably for thetreatment of a disease or condition associated with or characterized bythe expression of CD123 as further defined herein.

The disease or condition associated with or characterized by theexpression of CD123 may be cancer. For example, the cancer may beselected from the group consisting of: acute myeloid leukemia (AML),chronic myelogenous leukemia (CML), including blastic crisis of CML andAbelson oncogene associated with CML (Bcr-ABL translocation),myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL),chronic lymphocytic leukemia (CLL), including Richter's syndrome orRichter's transformation of CLL, hairy cell leukemia (HCL), blasticplasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphomas(NHL), including mantel cell leukemia (MCL), and small lymphocyticlymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt'slymphoma.

The disease or condition associated with or characterized by theexpression of CD123 may be an inflammatory condition. For example, theinflammatory condition may be selected from the group consisting of:Autoimmune Lupus (SLE), allergy, asthma and rheumatoid arthritis.

The invention additionally provides a pharmaceutical compositioncomprising any of the above-described diabodies and a physiologicallyacceptable carrier.

The invention additionally provides a use of the above-describedpharmaceutical composition in the treatment of a disease or conditionassociated with or characterized by the expression of CD123.

The invention is particularly directed to the embodiment of such use,wherein the disease or condition associated with or characterized by theexpression of CD123 is cancer (especially a cancer selected from thegroup consisting of: acute myeloid leukemia (AML), chronic myelogenousleukemia (CML), including blastic crisis of CML and Abelson oncogeneassociated with CML (Bcr-ABL translocation), myelodysplastic syndrome(MDS), acute B lymphoblastic leukemia (B-ALL), chronic lymphocyticleukemia (CLL), including Richter's syndrome or Richter's transformationof CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cellneoplasm (BPDCN), non-Hodgkin lymphomas (NHL), including mantel cellleukemia (MCL), and small lymphocytic lymphoma (SLL), Hodgkin'slymphoma, systemic mastocytosis, and Burkitt's lymphoma).

The invention is also particularly directed to the embodiment of suchuse, wherein the disease or condition associated with or characterizedby the expression of CD123 is an inflammatory condition (especially aninflammatory condition selected from the group consisting of: AutoimmuneLupus (SLE), allergy, asthma, and rheumatoid arthritis).

Terms such as “about” should be taken to mean within 10%, morepreferably within 5%, of the specified value, unless the contextrequires otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that CD123 was known to be expressed on leukemic stemcells.

FIG. 2 illustrates the structures of the first and second polypeptidechains of a two chain CD123×CD3 bi-specific monovalent diabody of thepresent invention.

FIGS. 3A and 3B illustrate the structures of two versions of the first,second and third polypeptide chains of a three chain CD123×CD3bi-specific monovalent Fc diabody of the present invention (Version 1,FIG. 3A; Version 2, FIG. 3B).

FIG. 4 (Panels A-E) shows the ability of different CD123×CD3 bi-specificdiabodies to mediate T cell redirected killing of target cellsdisplaying varying amount of CD123. The Figure provides dose-responsecurves indicating that the sequence-optimized CD123×CD3 bi-specificdiabody (“DART-A”) having an Albumin-Binding Domain (DART-A with ABD“w/ABD”) exhibited greater cytotoxicity than a control bi-specificdiabody (Control DART) or a non-sequence-optimized CD123×CD3 bi-specificdiabody (“DART-B”) in multiple target cell types: RS4-11 (Panel A); TF-1(Panel B); Molm-13 (Panel C); Kasumi-3 (Panel D); and THP-1 (Panel E) atan E:T (effector: target) ratio of 10:1.

FIG. 5 (Panels A-D) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A), sequence-optimized CD123×CD3bi-specific diabody having an Albumin-Binding Domain (DART-A with ABD“w/ABD”) and sequence-optimized CD123×CD3 bi-specific diabody having animmunoglobulin IgG Fc Domain (DART-A with Fc “w/Fc”) to mediate T cellactivation during redirected killing of target cells. The Figurepresents dose-response curves showing the cytotoxicity mediated byDART-A, DART-A w/ABD and DART-A w/Fc in Kasumi-3 (Panel A) and THP-1(Panel B) cells and purified CD8 T cells at an E:T (effector cell:targetcell) ratio of 10:1 (18 hour incubation). Panels C and D showdose-response curves of T cell activation using the marker CD25 on CD8 Tcells in the presence (Panel D) and absence (Panel C) of target cells.

FIG. 6 (Panels A-B) shows Granzyme B and Perforin levels in CD4 and CD8T cells after treatment with the sequence-optimized CD123×CD3bi-specific diabody (DART-A) (Panel A) or a control bi-specific diabody(Control DART) (Panel B) in the presence of Kasumi-3 target cells andresting T cells at an E:T ratio of 10:1.

FIG. 7 (Panels A-B) shows the in vivo antitumor activity of thesequence-optimized CD123×CD3 bi-specific diabody (DART-A) at nanogramper kilogram dosing levels. MOLM-13 cells (intermediate CD123expression) were co-mixed with T cells and implanted subcutaneously (T:E1:1) in NSG mice. Intravenous treatment was once daily for 8 days (QDx8)starting at implantation. Various concentrations of DART-A were comparedto a control bi-specific diabody (Control DART). Panel A shows theMolm-13 cells alone or with T cells, and the effect of various doses ofDART-A on tumor volume even to times beyond 30 days. Panel B shows theeffect of increasing doses of DART-A on tumor volume seen in NSG micereceiving MOLM-13 cells and T cells (T:E 1:1) for a time course of 0-18days.

FIG. 8 shows the in vivo antitumor activity of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) on RS4-11 cells (ALL withmonocytic features). Cells were co-mixed with T cells and implantedsubcutaneously (T:E 1:1) in NSG mice. Intravenous treatment was oncedaily for 4 days (QDx4) starting at implantation. Various concentrationsof DART-A were compared to a control bi-specific diabody (Control DART).

FIG. 9 (Panels A-B) shows CD123+ blasts in bone marrow mononucleocytes(BM MNC) and peripheral blood mononucleocytes (PBMCs) from AML patient 1(Panel A) compared to Kasumi-3 AML cell line (Panel B).

FIG. 10 (Panels A-C) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to mediate blast reduction inprimary AML at 120 h (Panel A), drive T cell expansion in primary AML at120 h (Panel B) and induce T cell activation in AML at 48 h and 72 h(Panel C).

FIG. 11 (Panels A-H) shows the identification of the CD123+ blastpopulation in a primary sample of ALL PBMCs. Panels A and E show theforward and side scatter of the input population of normal PBMC (PanelA) and ALL PBMCs (Panel E). Panels B and F show the identification ofthe lymphocyte population as primarily B cells (Panel B) and leukemicblast cells (Panel F). Panels C and G show identification of thepopulation of lymphocytes that are CD123+. Panels D and H show theidentification of CD19+ cells and CD123+ cells.

FIG. 12 (Panels A-B) shows the identification of the CD4 and CD8populations of T cells in a primary sample of ALL PBMCs. Panel A showsthe forward and side scatter of the input ALL PBMCs. Panel B shows theCD4 or CD8 populations of T cells present in the samples. The numbersindicate that CD4 T cells represent approximately 0.5% of the totalcells and CD8 T cells represent approximately 0.4% of the total cellspresent in the ALL PBMC sample.

FIG. 13 (Panels A-H) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to mediate ALL blast depletionwith autologous CTL. Panels A and E show the forward and side scatter ofthe input population of normal PBMC (Panel A) and ALL PBMCs (Panel E).The PBMCs were untreated (Panels B and F), treated with a controlbi-specific diabody (Control DART) (Panels C and G) or treated withDART-A (Panels D and H) and incubated for 7 days followed by stainingfor CD34 and CD19.

FIG. 14 (Panels A-L) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to mediate T cell expansion(Panels A, B, C, G, H and I) and activation (Panels D, E, F, J, K and L)in normal PBMC (Panels A-F) and ALL PBMC (Panels G-L). The cells wereuntreated (Panels A, D, G and J), or treated with a control bi-specificdiabody (Control DART) (Panels B, E, H and K) or DART-A (Panels C, F, Iand L) for 7 days.

FIG. 15 (Panels A-C) shows the identification of the AML blastpopulation and T cells in a primary AML sample. Panel A shows theforward and side scatter of the input AML PBMCs. Panel B shows theidentification of the AML blast population in the AML sample. Panel Cshows the identification of the T cell population in the AML sample.

FIG. 16 (Panels A-C) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to mediate AML blast depletionwith autologous CTL and T cell expansion. Primary AML PBMCs from patient2 were incubated with PBS, a control bi-specific diabody (Control DART)or DART-A for 144 h. Blast cells (Panel A), CD4 T cells (Panel B) andCD8 T cells (Panel C) were counted.

FIG. 17 (Panels A-D) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to mediate T cell activation inAML. CD25 (Panel A) and Ki-67 (Panel B) expression was determined forthe CD4 and CD8 T cells from AML patient 2 following incubation with acontrol bi-specific diabody (Control DART) or DART-A with autologousPBMCs. The level of perforin (Panel C) and granzyme B (Panel D) wasdetermined for the CD4 and CD8 T cells from AML patient 2 followingincubation with Control DART or DART-A with autologous PBMCs.

FIG. 18 (Panels A-D) shows that the sequence-optimized CD123×CD3bi-specific diabody (DART-A) is capable of cross-reacting with bothhuman and primate CD123 and CD3 proteins. The panels show BIACOREsensogram traces of the results of analyses conducted to assess theability of DART-A to bind to human (Panels A and C) and non-humanprimate (Panels B and D) CD3 (Panels A and B) and CD123 (Panels C and D)proteins. The KD values are provided.

FIG. 19 (Panels A-B) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to mediate autologous monocytedepletion in vitro with human and cynomolgus monkey PBMCs. The Panelspresent the results of dose-response curves of DART-A-mediatedcytotoxicity with primary human PBMCs (Panel A) or cynomolgus monkeyPBMCs (Panel B).

FIG. 20 (Panels A-N) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to mediate the depletion of pDCin cynomolgus monkeys without systemic cytokine induction. Panels A-Dshow control results obtained at 4 h and 4 d with vehicle and carrier.Panels E-H show control results obtained at 4 h and 4 d with a controlbi-specific diabody (Control DART). Panels I-N show results obtained at4 h and 4 d at 10 ng/kg/d and at 4 d with 30 ng/kg/d of DART-A.

FIG. 21 (Panels A-D) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to mediate dose-dependentdepletion of pDC in cynomolgus monkeys. Cynomolgus monkeys were dosedwith DART-A at 0.1, 1, 10, 30 100, 300, or 1000 ng/kg. PBMCs wereevaluated at the indicated time and total B cells (Panel A), monocytes(Panel B), NK cells (Panel C) and pDC (Panel D) were counted.

FIG. 22 (Panels A-D) shows the ability of the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) to intermittently modulate Tcells in cynomolgus monkeys. Cynomolgus monkeys were dosed with DART-Aat 0.1, 1, 10, 30 100, 300, or 1000 ng/kg. PBMCs were evaluated at theindicated time and total T cells (Panel A), CD4 T cells (Panel B), CD69cells (Panel C) and CD8 T cells (Panel D) were counted.

FIG. 23 shows the SDS-PAGE analysis of purified DART-A protein underreducing (left) and non-reducing (right) conditions.

FIGS. 24A-24B show the physicochemical characterization of purifiedDART-A. FIG. 24A: SEC profile of DART-A protein on a calibrated TSKG3000SWxL column. FIG. 24B: Mass spectrum of DART-A protein.

FIGS. 25A-25D show SPR analysis of DART-A binding to immobilized humanor cynomolgus monkey CD123 and CD3. Dashed lines represent the globalfit to a 1:1 Langmuir model of the experimental binding curves obtainedat DART-A concentrations of 0, 6.25, 12.5, 25, 50 or 100 nM (continuouslines). The data are representative of three independent experiments.

FIGS. 26A-26E show that DART-A was capable of simultaneously bindingboth CD3 and CD123. FIG. 26A-26B provides the results of a bifunctionalELISA and demonstrates simultaneous engagement of both target antigensof DART-A. ELISA plates were coated with human CD123 (FIG. 26A) orcynomolgus monkey CD123 (FIG. 26B). Titrating DART-A and Control DARTconcentrations were followed by detection with human CD3-biotin. FIGS.26C-26E demonstrate cell-surface binding of DART-A on CD123+ Molm-13target cell (FIG. 26C), human T cells (FIG. 26D) and cynomolgus T cells(FIG. 26E). Binding was detected by FACS analysis using a monoclonalantibody specific to E-coil and K-coil region of the DART-A or ControlDART molecule.

FIGS. 27A-27H show the ability of DART-A to mediate redirected targetcell killing by human or monkey effector cells against CD123+ Kasumi-3leukemic cell lines, demonstrate the ability of the molecules to bind tosubsets of normal circulating leukocytes, including pDCs and monocytesand demonstrate the ability of the molecules to depleteCD14⁻CD123^(high) cells (pDC and basophils) without affecting monocytes(CD14+ cells). FIG. 27A shows the relative anti-CD123-PE binding siteson U937 and Kasumi-3 leukemic cell lines as determined by QFACSanalysis.

FIG. 27B shows the relatively low percent cytotoxicity mediated byDART-A or Control DART on an AML cell line (U937 cells) which, as shownin FIG. 27A have relatively few CD123 binding sites). FIG. 27C shows thepercent cytotoxicity mediated by DART-A or Control DART in the presenceof purified human T cells (as effector cells) on an AML cell line(Kasumi-3 cells) which, as shown in FIG. 27A have a substantial numberof CD123 binding sites. In FIGS. 27B-27C, the E:T ratio is 10:1. FIG.27D shows the percent cytotoxicity mediated by DART-A or Control DART inthe presence of purified cynomolgus monkey PBMCs (as effector cells) onKasumi-3 cells (the E:T ratio is 15:1), and demonstrates that DART-A canbind cynomolgus monkey T cells. FIG. 27E shows the relativeanti-CD123-PE binding sites on Kasumi-3 cells, human monocytes, humanplasmacytoid dendritic cells (“pDC”), cynomolgus monkey monocytes andcynomolgus monkey plasmacytoid dendritic cells as determined by QFACSanalysis. FIG. 27F shows the ability of DART-A to depleteCD14⁻CD123^(lo) cells. FIG. 27G shows the ability of DART-A to depletehuman CD14⁻CD123^(Hi) cells. FIG. 27H shows the ability of DART-A todeplete cynomolgus monkey CD14⁻CD123^(Hi) cells. Cytotoxicity wasdetermined by LDH release, with EC50 values determined using GraphPadPRISM® software.

FIG. 28 shows the use of a two-compartment model to estimatepharmacokinetic parameters of DART-A. The data show the end of infusion(EOI) serum concentrations of DART-A in cynomolgus monkeys afterreceiving a 96-hour infusion at 100 ng/kg/day 300 ng/kg/day, 600ng/kg/day, and 1000 ng/kg/day Dose. Each point represents an individualanimal; horizontal lines represent the mean value for the dose group.

FIGS. 29A-29C show the effect of DART-A infusions on the production ofthe cytokine, IL-6. Serum IL-6 levels (mean±SEM) in monkeys infused withDART-A are shown by treatment group. Cynomolgus monkeys were treatedwith vehicle control on Day 1, followed by 4 weekly infusions of eithervehicle (Group 1) (FIG. 29A) or DART-A administered as 4-day weeklyinfusions starting on Days 8, 15, 22, and 29 (Groups 2-5) (FIG. 29B) oras a 7-day/week infusion for 4 weeks starting on Days 8 (Group 6) (FIG.29C). Treatment intervals are indicated by the filled gray bars.

FIGS. 30A-30F show the effect of DART-A infusions on the depletion ofCD14-/CD123+ cells (FIGS. 30A-30C) and CD303+ cells (FIGS. 30D-30F). Themean±SEM of the circulating levels of CD14-/CD123+(FIGS. 30A-30C) orCD303+(FIGS. 30D-30F) by Study Day and by group is shown. Cynomolgusmonkeys were treated with vehicle control on Day 1, followed by 4 weeklyinfusions of either vehicle (Group 1) (FIGS. 30A and 30D) or DART-Aadministered as 4-day weekly infusions starting on Days 8, 15, 22, and29 (Groups 2-5) (FIGS. 30A and 30E) or as a 7-day/week infusion for 4weeks starting on Days 8 (Group 6) (FIGS. 30C and 30F). Treatmentintervals are indicated by the filled gray bars.

FIGS. 31A-31I show the observed changes in T cell populations (FIGS.31A-31C), CD4+ cell populations (FIGS. 31D-31F) and CD8+ cellpopulations (FIGS. 31G-31I) receiving DART-A administered as 4-dayinfusions starting on Days 8, 15, 22, and 29. Legend: CD25+ (graysquares); CD69+ (gray triangles), PD-1+ (white triangles); Tim-3+ (whitesquares). T cells were enumerated via the CD4 and CD8 markers, ratherthan the canonical CD3, to eliminate possible interference the DART-A.Cynomolgus monkeys were treated with vehicle control on Day 1, followedby 4 weekly infusions of either vehicle (Group 1) or DART-A administeredas 4-day weekly infusions starting on Days 8, 15, 22, and 29 (Group 5)or as a 7-day/week infusion for 4 weeks starting on Days 8 (Group 6).Treatment intervals are indicated by the filled gray bars. The mean±SEMof the absolute number of total circulating T cells by Study Day andgroup is shown (FIGS. 31A-31C). Relative values (mean percent±SEM) ofCD25+, CD69+, PD-1+ and Tim-3+ of CD4 (FIGS. 31D-31E) or CD8 T cells(FIGS. 31F-31H) by Study Day and by group is shown.

FIGS. 32A-32F show the observed changes in T CD4+ cell populations(FIGS. 32A-32C) and CD8+ cell populations (FIGS. 32D-32F) during andafter a continuous 7-day infusion of DART-A. The mean±SEM percent ofCD25+, CD69+, PD-1+ and Tim-3+ on CD4 (FIGS. 32A-32C) or CD8 (FIGS.32D-32F) T cells by Study Day for Groups 2, 3 and 4 are shown. Treatmentintervals are indicated by the filled gray bars. Legend: CD25+ (graysquares); CD69+ (gray triangles), PD-1+ (white triangles); Tim-3+ (whitesquares).

FIGS. 33A-33F show the observed changes in T CD4+ cell populations(FIGS. 33A-33C) and CD8+ cell populations (FIGS. 33D-33F) during andafter a continuous 7-day infusion of DART-A. The mean±SEM percent ofCD4+Naïve (CD95-/CD28+), CMT (CD95+/CD28+), and EMT (CD95+/CD28-) Tcells in CD4+ population (FIGS. 33A-33C) or CD8 population (FIGS.33D-33F) by Study Day for Groups 2, 3 and 4 are shown. Cynomolgusmonkeys were treated with vehicle control on Day 1, followed by 4 weeklyinfusions of or DART-A administered as 4-day weekly infusions startingon Days 8, 15, 22, and 29 (Groups 2-4). Treatment intervals areindicated by the filled gray bars. Legend: Naïve (white triangles); CMT(black triangles), EMT (gray squares).

FIG. 34 shows DART-A-mediated cytotoxicity against Kasumi-3 cells withPBMCs from either naïve monkeys or monkeys treated with multipleinfusions of DART-A.

FIGS. 35A-35F show that DART-A exposure increased the relative frequencyof central memory CD4 cells and effector memory CD8+ cells at theexpense of the corresponding naïve T cell population. The mean±SEMpercent of CD4+ Naïve (CD95−/CD28+), CMT (CD95+/CD28+), and EMT(CD95+/CD28−) T cells in CD4+ population (FIGS. 35A-35C) or in CD8+population (FIGS. 35D-35F) by Study Day and by Group is shown.Cynomolgus monkeys were treated with vehicle control on Day 1, followedby 4 weekly infusions of either vehicle (Group 1) or DART-A administeredas 4-day weekly infusions starting on Days 8, 15, 22, and 29 (Group 5)or as a 7-day/week infusion for 4 weeks starting on Days 8 (Group 6).Treatment intervals are indicated by the filled gray bars. Legend: Naïve(white triangles); CMT (black triangles), EMT (gray squares).

FIGS. 36A-36F show the effect of DART-A on red cell parameters inmonkeys that had received infusions of the molecules. Circulating RBCs(FIGS. 36A-36C) or reticulocytes (FIGS. 36D-36F) levels (mean±SEM) insamples collected at the indicated time points from monkeys treated withDART-A are shown.

FIGS. 37A-37B show that the frequency (mean percent±SEM) of CD123+ cells(FIG. 37A) or HSC (CD34+/CD38−/CD45−/CD90+ cells) (FIG. 37B) within theLin− cell population in bone marrow samples collected at the indicatedtime points from monkeys treated with DART-A. Cynomolgus monkeys weretreated with vehicle control on Day 1, followed by 4 weekly infusions ofeither vehicle (Group 1) or DART-A administered as 4-day weeklyinfusions starting on Days 8, 15, 22, and 29 (Groups 2-5) or as a7-day/week infusion for 4 weeks starting on Days 8 (Group 6).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to sequence-optimized CD123×CD3bi-specific monovalent diabodies that are capable of simultaneousbinding to CD123 and CD3, and to the uses of such molecules in thetreatment of hematologic malignancies. Although non-optimized CD123×CD3bi-specific diabodies are fully functional, analogous to theimprovements obtained in gene expression through codon optimization(see, e.g., Grosjean, H. et al. (1982) “Preferential Codon Usage InProkaryotic Genes: The Optimal Codon Anticodon Interaction Energy AndThe Selective Codon Usage In Efficiently Expressed Genes” Gene18(3):199-209), it is possible to further enhance the stability and/orfunction of CD123×CD3 bi-specific diabodies by modifying or refiningtheir sequences.

The preferred CD123×CD3 bi-specific diabodies of the present inventionare composed of at least two polypeptide chains that associate with oneanother to form one binding site specific for an epitope of CD123 andone binding site specific for an epitope of CD3 (FIG. 2). The individualpolypeptide chains of the diabody are covalently bonded to one another,for example by disulfide bonding of cysteine residues located withineach polypeptide chain. Each polypeptide chain contains an AntigenBinding Domain of a Light Chain Variable Domain, an Antigen BindingDomain of a Heavy Chain Variable Domain and a heterodimerization domain.An intervening linker peptide (Linker 1) separates the Antigen BindingDomain of the Light Chain Variable Domain from the Antigen BindingDomain of the Heavy Chain Variable Domain. The Antigen Binding Domain ofthe Light Chain Variable Domain of the first polypeptide chain interactswith the Antigen Binding Domain of the Heavy Chain Variable Domain ofthe second polypeptide chain in order to form a first functional antigenbinding site that is specific for the first antigen (i.e., either CD123or CD3). Likewise, the Antigen Binding Domain of the Light ChainVariable Domain of the second polypeptide chain interacts with theAntigen Binding Domain of the Heavy Chain Variable Domain of the firstpolypeptide chain in order to form a second functional antigen bindingsite that is specific for the second antigen (i.e., either CD123 or CD3,depending upon the identity of the first antigen). Thus, the selectionof the Antigen Binding Domain of the Light Chain Variable Domain and theAntigen Binding Domain of the Heavy Chain Variable Domain of the firstand second polypeptide chains are coordinated, such that the twopolypeptide chains collectively comprise Antigen Binding Domains oflight and Heavy Chain Variable Domains capable of binding to CD123 andCD3.

The formation of heterodimers of the first and second polypeptide chainscan be driven by the heterodimerization domains. Such domains includeGVEPKSC (SEQ ID NO:50) (or VEPKSC; SEQ ID NO:51) on one polypeptidechain and GFNRGEC (SEQ ID NO:52) (or FNRGEC; SEQ ID NO:53) on the otherpolypeptide chain (US2007/0004909). Alternatively, such domains can beengineered to contain coils of opposing charges. The heterodimerizationdomain of one of the polypeptide chains comprises a sequence of at leastsix, at least seven or at least eight positively charged amino acids,and the heterodimerization domain of the other of the polypeptide chainscomprises a sequence of at least six, at least seven or at least eightnegatively charged amino acids. For example, the first or the secondheterodimerization domain will preferably comprise a sequence of eightpositively charged amino acids and the other of the heterodimerizationdomains will preferably comprise a sequence of eight negatively chargedamino acids. The positively charged amino acid may be lysine, arginine,histidine, etc. and/or the negatively charged amino acid may be glutamicacid, aspartic acid, etc. The positively charged amino acid ispreferably lysine and/or the negatively charged amino acid is preferablyglutamic acid.

The CD123×CD3 bi-specific diabodies of the present invention areengineered so that such first and second polypeptide chains covalentlybond to one another via cysteine residues along their length. Suchcysteine residues may be introduced into the intervening linker thatseparates the VL and VH domains of the polypeptides. Alternatively, andmore preferably, a second peptide (Linker 2) is introduced into eachpolypeptide chain, for example, at the amino-terminus of the polypeptidechains or a position that places Linker 2 between the heterodimerizationdomain and the Antigen Binding Domain of the Light Chain Variable Domainor Heavy Chain Variable Domain.

In particular embodiments, the sequence-optimized CD123×CD3 bi-specificmonovalent diabodies of the present invention further have animmunoglobulin Fc Domain or an Albumin-Binding Domain to extendhalf-life in vivo.

The CD123×CD3 bi-specific monovalent diabodies of the present inventionthat comprise an immunoglobulin Fc Domain (i.e., CD123×CD3 bi-specificmonovalent Fc diabodies) are composed of a first polypeptide chain, asecond polypeptide chain and a third polypeptide chain. The first andsecond polypeptide chains associate with one another to form one bindingsite specific for an epitope of CD123 and one binding site specific foran epitope of CD3. The first polypeptide chain and the third polypeptidechain associate with one another to form an immunoglobulin Fc Domain(FIG. 3A and FIG. 3B). The first and second polypeptide chains of thebi-specific monovalent Fc diabody are covalently bonded to one another,for example by disulfide bonding of cysteine residues located withineach polypeptide chain.

The first and third polypeptide chains are covalently bonded to oneanother, for example by disulfide bonding of cysteine residues locatedwithin each polypeptide chain. The first and second polypeptide chainseach contain an Antigen Binding Domain of a Light Chain Variable Domain,an Antigen Binding Domain of a Heavy Chain Variable Domain and aheterodimerization domain. An intervening linker peptide (Linker 1)separates the Antigen Binding Domain of the Light Chain Variable Domainfrom the Antigen Binding Domain of the Heavy Chain Variable Domain. TheAntigen Binding Domain of the Light Chain Variable Domain of the firstpolypeptide chain interacts with the Antigen Binding Domain of the HeavyChain Variable Domain of the second polypeptide chain in order to form afirst functional antigen binding site that is specific for the firstantigen (i.e., either CD123 or CD3). Likewise, the Antigen BindingDomain of the Light Chain Variable Domain of the second polypeptidechain interacts with the Antigen Binding Domain of the Heavy ChainVariable Domain of the first polypeptide chain in order to form a secondfunctional antigen binding site that is specific for the second antigen(i.e., either CD3 or CD123, depending upon the identity of the firstantigen). Thus, the selection of the Antigen Binding Domain of the LightChain Variable Domain and the Antigen Binding Domain of the Heavy ChainVariable Domain of the first and second polypeptide chains arecoordinated, such that the two polypeptide chains collectively compriseAntigen Binding Domains of light and Heavy Chain Variable Domainscapable of binding to CD123 and CD3. The first and third polypeptidechains each contain some or all of the CH2 Domain and/or some or all ofthe CH3 Domain of a complete immunoglobulin Fc Domain and acysteine-containing peptide. The some or all of the CH2 Domain and/orthe some or all of the CH3 Domain associate to form the immunoglobulinFc Domain of the bi-specific monovalent Fc diabodies of the presentinvention. The first and third polypeptide chains of the bi-specificmonovalent Fc diabodies of the present invention are covalently bondedto one another, for example by disulfide bonding of cysteine residueslocated within the cysteine-containing peptide of the polypeptidechains.

I. The Sequence-Optimized CD123×CD3 Bi-Specific Diabody, “DART-A”

The invention provides a sequence-optimized bi-specific diabody capableof simultaneously and specifically binding to an epitope of CD123 and toan epitope of CD3 (a “CD123×CD3” bi-specific diabody or DART-A). Asdiscussed below, DART-A was found to exhibit enhanced functionalactivity relative to other non-sequence-optimized CD123×CD3 bi-specificdiabodies of similar composition, and is thus termed a“sequence-optimized” CD123×CD3 bi-specific diabody.

The sequence-optimized CD123×CD3 bi-specific diabody (DART-A) comprisesa first polypeptide chain and a second polypeptide chain. The firstpolypeptide chain of the bi-specific diabody will comprise, in theN-terminal to C-terminal direction, an N-terminus, a Light ChainVariable Domain (VL Domain) of a monoclonal antibody capable of bindingto CD3 (VL_(CD3)), an intervening linker peptide (Linker 1), a HeavyChain Variable Domain (VH Domain) of a monoclonal antibody capable ofbinding to CD123 (VH_(CD123)), and a C-terminus. A preferred sequencefor such a VL_(CD3) Domain is SEQ ID NO:21:

QAVVTQEPSLTVSPGGIVTLICRSSTGAVITSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVF GGGTKLTVLG

The Antigen Binding Domain of VL_(CD3) comprises CDR1 SEQ ID NO:38:RSSTGAVTTSNYAN, CDR2 SEQ ID NO:39: GTNKRAP, and CDR3 SEQ ID NO:40:ALWYSNLWV.

A preferred sequence for such Linker 1 is SEQ ID NO:29: GGGSGGGG. Apreferred sequence for such a VH_(CD123) Domain is SEQ ID NO:26:

EVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSH LLRASWFAYWGQGTLVTVSS

The Antigen Binding Domain of VH_(CD123) comprises CDR1 SEQ ID NO:47:DYYMK, CDR2 SEQ ID NO:48: DIIPSNGATFYNQKFKG, and CDR3 SEQ ID NO:49:SHLLRAS.

The second polypeptide chain will comprise, in the N-terminal toC-terminal direction, an N-terminus, a VL domain of a monoclonalantibody capable of binding to CD123 (VL_(CD123)), an intervening linkerpeptide (e.g., Linker 1), a VH domain of a monoclonal antibody capableof binding to CD3 (VH_(CD3)), and a C-terminus. A preferred sequence forsuch a VL_(CD123) Domain is SEQ ID NO:25:

DFVMTQSPDSLAVSLGERVIMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFILTISSLQAEDVAVYYCQNDYSY PYTFGQGTKLEIK

The Antigen Binding Domain of VL_(CD123) comprises CDR1 SEQ ID NO:44:KSSQSLLNSGNQKNYLT, CDR2 SEQ ID NO:45: WASTRES, and CDR3 SEQ ID NO:46:QNDYSYPYT.

A preferred sequence for such a VH_(CD3) Domain is SEQ ID NO:22:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

The Antigen Binding Domain of VH_(CD3) comprises CDR1 SEQ ID NO:41:TYAMN, CDR2 SEQ ID NO:42: RIRSKYNNYATYYADSVKD, and CDR3 SEQ ID NO:43:HGNFGNSYVSWFAY.

The sequence-optimized CD123×CD3 bi-specific diabodies of the presentinvention are engineered so that such first and second polypeptidescovalently bond to one another via cysteine residues along their length.Such cysteine residues may be introduced into the intervening linker(e.g., Linker 1) that separates the VL and VH domains of thepolypeptides. Alternatively, and more preferably, a second peptide(Linker 2) is introduced into each polypeptide chain, for example, at aposition N-terminal to the VL domain or C-terminal to the VH domain ofsuch polypeptide chain. A preferred sequence for such Linker 2 is SEQ IDNO:30: GGCGGG.

The formation of heterodimers can be driven by further engineering suchpolypeptide chains to contain polypeptide coils of opposing charge.Thus, in a preferred embodiment, one of the polypeptide chains will beengineered to contain an “E-coil” domain (SEQ ID NO:34:EVAALEKEVAALEKEVAALEKEVAALEK) whose residues will form a negative chargeat pH 7, while the other of the two polypeptide chains will beengineered to contain an “K-coil” domain (SEQ ID NO:35:KVAALKEKVAALKEKVAALKEKVAALKE) whose residues will form a positive chargeat pH 7. The presence of such charged domains promotes associationbetween the first and second polypeptides, and thus fostersheterodimerization.

It is immaterial which coil is provided to the first or secondpolypeptide chains. However, a preferred sequence-optimized CD123×CD3bi-specific diabody of the present invention (“DART-A”) has a firstpolypeptide chain having the sequence (SEQ ID NO:1):

QAVVTQEPSLTVSPGGIVTLICRSSTGAVITSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGEVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEK

DART-A Chain 1 is composed of: SEQ ID NO:21—SEQ ID NO:29—SEQ IDNO:26—SEQ ID NO:30—SEQ ID NO:34. A DART-A Chain 1 encodingpolynucleotide is SEQ ID NO:2:

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgaccctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattgggtgcagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaaagggctccctggacccctgcacggttttctggaagtctgctgggcggaaaggccgctctgactattaccggggcacaggccgaggacgaagccgattactattgtgctctgtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctgggagggggtggatccggcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtgaaggtgtcttgcaaagccagtggctacaccttcacagactactatatgaagtgggtcaggcaggctccaggacagggactggaatggatcggcgatatcattccttccaacggggccactttctacaatcagaagtttaaaggcagggtgactattaccgtggacaaatcaacaagcactgcttatatggagctgagctccctgcgctctgaagatacagccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattggggacagggcaccctggtgacagtgtcttccggaggatgtggcggtggagaagtggccgcactggagaaagaggttgctgctttggagaaggaggtcgctgcacttgaaaaggaggt cgcagccctggagaaa

The second polypeptide chain of DART-A has the sequence (SEQ ID NO:3):

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

DART-A Chain 2 is composed of: SEQ ID NO:25—SEQ ID NO:29—SEQ IDNO:22—SEQ ID NO:30—SEQ ID NO:35. A DART-A Chain 2 encodingpolynucleotide is SEQ ID NO:4:

gacttcgtgatgacacagtctcctgatagtctggccgtgagtctgggggagcgggtgactatgtcttgcaagagctcccagtcactgctgaacagcggaaatcagaaaaactatctgacctggtaccagcagaagccaggccagccccctaaactgctgatctattgggcttccaccagggaatctggcgtgcccgacagattcagcggcagcggcagcggcacagattttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactattgtcagaatgattacagctatccctacactttcggccaggggaccaagctggaaattaaaggaggcggatccggcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtccagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagcacatacgctatgaattgggtccgccaggctccagggaaggggctggagtgggttggaaggatcaggtccaagtacaacaattatgcaacctactatgccgactctgtgaaggatagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaattcttacgtgtcttggtttgcttattggggacaggggacactggtgactgtgtcttccggaggatgtggcggtggaaaagtggccgcactgaaggagaaagttgctgctttgaaagagaaggtcgccgcacttaaggaaaaggtcgcagccctgaaagag

As discussed below, the sequence-optimized CD123×CD3 bi-specific diabody(DART-A) was found to have the ability to simultaneously bind CD123 andCD3 as arrayed by human and monkey cells. Provision of DART-A was foundto cause T cell activation, to mediate blast reduction, to drive T cellexpansion, to induce T cell activation and to cause the redirectedkilling of target cancer cells.

II. Comparative Non-Sequence-Optimized CD123×CD3 Bi-Specific Diabody,“DART-B”

DART-B is a non-sequence-optimized CD123×CD3 bi-specific diabody havinga gross structure that is similar to that of DART-A. The firstpolypeptide chain of DART-B will comprise, in the N-terminal toC-terminal direction, an N-terminus, a VL domain of a monoclonalantibody capable of binding to CD3 (VL_(CD3)), an intervening linkerpeptide (Linker 1), a VH domain of a monoclonal antibody capable ofbinding to CD123 (VH_(CD123)), an intervening Linker 2, an E-coilDomain, and a C-terminus. The VL_(CD3) Domain of the first polypeptidechain of DART-B has the sequence (SEQ ID NO:23):

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAG TKLELKThe VH_(CD123) Domain of the first polypeptide chain of DART-B has thesequence (SEQ ID NO:28):

QVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSH LLRASWFAYWGQGTLVTVSS

Thus, DART-B Chain 1 is composed of: SEQ ID NO:23—SEQ ID NO:29—SEQ IDNO:28—SEQ ID NO:30—SEQ ID NO:34. The sequence of the first polypeptidechain of DART-B is (SEQ ID NO:5):

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGSGGGGQVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGEVAALEKEVA ALEKEVAALEKEVAALEK

A DART-B Chain 1 encoding polynucleotide is SEQ ID NO:6:

gacattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaaggaggcggatccggcggcggaggccaggtgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtgaaggtgtcttgcaaagccagtggctacaccttcacagactactatatgaagtgggtcaggcaggctccaggacagggactggaatggatcggcgatatcattccttccaacggggccactttctacaatcagaagtttaaaggcagggtgactattaccgtggacaaatcaacaagcactgcttatatggagctgagctccctgcgctctgaagatacagccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattggggacagggcaccctggtgacagtgtcttccggaggatgtggcggtggagaagtggccgcactggagaaagaggttgctgctttggagaaggaggtcgctgcacttgaaaaggaggtcgcagccctgga gaaa

The second polypeptide chain of DART-B will comprise, in the N-terminalto C-terminal direction, an N-terminus, a VL domain of a monoclonalantibody capable of binding to CD123 (VL_(CD23)), an intervening linkerpeptide (Linker 1) and a VH domain of a monoclonal antibody capable ofbinding to CD3 (VH_(CD3)), an intervening Linker 2, a K-coil Domain, anda C-terminus.

The VL_(CD23) Domain of the second polypeptide chain of DART-B has thesequence (SEQ ID NO:27):

DFVMTQSPDSLAVSLGERVIMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFILTISSLQAEDVAVYYCQNDYSY PYTFGQGTKLEIKThe VH_(CD3) Domain of the second polypeptide chain of DART-B has thesequence (SEQ ID NO:24):

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSS

Thus, DART-B Chain 2 is composed of: SEQ ID NO:27—SEQ ID NO:29—SEQ IDNO:24—SEQ ID NO:30—SEQ ID NO:35. The sequence of the second polypeptidechain of DART-B is (SEQ ID NO:7):

DFVMTQSPDSLAVSLGERVIMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFILTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGGSGGGGDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLIVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

A DART-B Chain 2 encoding polynucleotide is SEQ ID NO:8:

gacttcgtgatgacacagtctcctgatagtctggccgtgagtctgggggagcgggtgactatgtcttgcaagagctcccagtcactgctgaacagcggaaatcagaaaaactatctgacctggtaccagcagaagccaggccagccccctaaactgctgatctattgggcttccaccagggaatctggcgtgcccgacagattcagcggcagcggcagcggcacagattttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactattgtcagaatgattacagctatccctacactttcggccaggggaccaagctggaaattaaaggaggcggatccggcggcggaggcgatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctccggaggatgtggcggtggaaaagtggccgcactgaaggagaaagttgctgctttgaaagagaaggtcgccgcacttaaggaaaaggtcgcagccctgaaagag

III. Modified Variants of Sequence-Optimized CD123×CD3 Bi-SpecificDiabody (DART-A)

A. Sequence-Optimized CD123×CD3 Bi-Specific Diabody Having anAlbumin-Binding Domain (DART-A with ABD “w/ABD”)

In a second embodiment of the invention, the sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) will comprise one or moreAlbumin-Binding Domain (“ABD”) (DART-A with ABD “w/ABD”) on one or bothof the polypeptide chains of the diabody.

As disclosed in WO 2012/018687, in order to improve the in vivopharmacokinetic properties of diabodies, the diabodies may be modifiedto contain a polypeptide portion of a serum-binding protein at one ormore of the termini of the diabody. Most preferably, such polypeptideportion of a serum-binding protein will be installed at the C-terminusof the diabody. A particularly preferred polypeptide portion of aserum-binding protein for this purpose is the Albumin-Binding Domain(ABD) from streptococcal protein G. The Albumin-Binding Domain 3 (ABD3)of protein G of Streptococcus strain G148 is particularly preferred.

The Albumin-Binding Domain 3 (ABD3) of protein G of Streptococcus strainG148 consists of 46 amino acid residues forming a stable three-helixbundle and has broad albumin-binding specificity (Johansson, M. U. etal. (2002) “Structure, Specificity, And Mode Of Interaction ForBacterial Albumin-Binding Modules,” J. Biol. Chem. 277(10):8114-8120).Albumin is the most abundant protein in plasma and has a half-life of 19days in humans. Albumin possesses several small molecule binding sitesthat permit it to non-covalently bind to other proteins and therebyextend their serum half-lives.

Thus, the first polypeptide chain of such a sequence-optimized CD123×CD3bi-specific diabody having an Albumin-Binding Domain contains a thirdlinker (Linker 3), which separates the E-coil (or K-coil) of suchpolypeptide chain from the Albumin-Binding Domain. A preferred sequencefor such Linker 3 is SEQ ID NO:31: GGGS. A preferred Albumin-BindingDomain (ABD) has the sequence (SEQ ID NO:36):LAEAKVLANRELDKYGVSDYYKNLIDNAKSAEGVKALIDEILAALP.

Thus, a preferred first chain of a sequence-optimized CD123×CD3bi-specific diabody having an Albumin-Binding Domain has the sequence(SEQ ID NO:9):

QAVVTQEPSLTVSPGGIVTLICRSSTGAVITSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGEVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGGGSLAEAKVLANRELDKYGVSDYYKNLIDNAKSAEGVKALIDEILAALP

A sequence-optimized CD123×CD3 diabody having an Albumin-Binding Domainis composed of: SEQ ID NO:21—SEQ ID NO:29—SEQ ID NO:26—SEQ ID NO:30—SEQID NO:34—SEQ ID NO:31—SEQ ID NO:36. A polynucleotide encoding such asequence-optimized CD123×CD3 diabody having an Albumin-Binding Domainderivative is SEQ ID NO:10:

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgaccctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattgggtgcagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaaagggctccctggacccctgcacggttttctggaagtctgctgggcggaaaggccgctctgactattaccggggcacaggccgaggacgaagccgattactattgtgctctgtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctgggagggggtggatccggcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtgaaggtgtcttgcaaagccagtggctacaccttcacagactactatatgaagtgggtcaggcaggctccaggacagggactggaatggatcggcgatatcattccttccaacggggccactttctacaatcagaagtttaaaggcagggtgactattaccgtggacaaatcaacaagcactgcttatatggagctgagctccctgcgctctgaagatacagccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattggggacagggcaccctggtgacagtgtcttccggaggatgtggcggtggagaagtggccgcactggagaaagaggttgctgctttggagaaggaggtcgctgcacttgaaaaggaggtcgcagccctggagaaaggcggcgggtctctggccgaagcaaaagtgctggccaaccgcgaactggataaatatggcgtgagcgattattataagaacctgattgacaacgcaaaatccgcggaaggcgtgaaagcactgattgatgaaat tctggccgccctgcct

The second polypeptide chain of such a sequence-optimized CD123×CD3diabody having an Albumin-Binding Domain has the sequence describedabove (SEQ ID NO:3) and is encoded by a polynucleotide having thesequence of SEQ ID NO:4.

B. Sequence-Optimized CD123×CD3 Bi-Specific Diabodies Having an IgG FcDomain (DART-A with Fc “w/Fc”)

In a third embodiment, the invention provides a sequence-optimizedCD123×CD3 bi-specific diabody composed of three polypeptide chains andpossessing an IgG Fc Domain (DART-A with Fc “w/Fc” Version 1 and Version2) (FIG. 3A-3B).

In order to form such IgG Fc Domain, the first and third polypeptidechain of the diabodies contain, in the N-terminal to C-terminaldirection, a cysteine-containing peptide, (most preferably, Peptide 1having the amino acid sequence (SEQ ID NO:55): DKTHTCPPCP), some or allof the CH2 Domain and/or some or all of the CH3 Domain of a completeimmunoglobulin Fc Domain, and a C-terminus. The some or all of the CH2Domain and/or the some or all of the CH3 Domain associate to form theimmunoglobulin Fc Domain of the bi-specific monovalent FcDomain-containing diabodies of the present invention. The first andsecond polypeptide chains of the bi-specific monovalent Fc diabodies ofthe present invention are covalently bonded to one another, for exampleby disulfide bonding of cysteine residues located within thecysteine-containing peptide of the polypeptide chains.

The CH2 and/or CH3 Domains of the first and third polypeptides need notbe identical, and advantageously are modified to foster complexingbetween the two polypeptides. For example, an amino acid substitution(preferably a substitution with an amino acid comprising a bulky sidegroup forming a ‘knob’, e.g., tryptophan) can be introduced into the CH2or CH3 Domain such that steric interference will prevent interactionwith a similarly mutated domain and will obligate the mutated domain topair with a domain into which a complementary, or accommodating mutationhas been engineered, i.e., ‘the hole’ (e.g., a substitution withglycine). Such sets of mutations can be engineered into any pair ofpolypeptides comprising the bi-specific monovalent Fc diabody molecule,and further, engineered into any portion of the polypeptides chains ofsaid pair. Methods of protein engineering to favor heterodimerizationover homodimerization are well known in the art, in particular withrespect to the engineering of immunoglobulin-like molecules, and areencompassed herein (see e.g., Ridgway et al. (1996) “Knobs-Into-Holes'Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization,”Protein Engr. 9:617-621, Atwell et al. (1997) “Stable Heterodimers FromRemodeling The Domain Interface Of A Homodimer Using A Phage DisplayLibrary,” J. Mol. Biol. 270: 26-35, and Xie et al. (2005) “A New FormatOf Bispecific Antibody: Highly Efficient Heterodimerization, ExpressionAnd Tumor Cell Lysis,” J. Immunol. Methods 296:95-101; each of which ishereby incorporated herein by reference in its entirety). Preferably the‘knob’ is engineered into the CH2-CH3 Domains of the first polypeptidechain and the ‘hole’ is engineered into the CH2-CH3 Domains of the thirdpolypeptide chain. Thus, the ‘knob’ will help in preventing the firstpolypeptide chain from homodimerizing via its CH2 and/or CH3 Domains. Asthe third polypeptide chain preferably contains the ‘hole’ substitutionit will heterodimerize with the first polypeptide chain as well ashomodimerize with itself A preferred knob is created by modifying anative IgG Fc Domain to contain the modification T366W. A preferred holeis created by modifying a native IgG Fc Domain to contain themodification T366S, L368A and Y407V. To aid in purifying the thirdpolypeptide chain homodimer from the final bi-specific monovalent Fcdiabody comprising the first, second and third polypeptide chains, theprotein A binding site of the CH2 and CH3 Domains of the thirdpolypeptide chain is preferably mutated by amino acid substitution atposition 435 (H435R). Thus, the third polypeptide chain homodimer willnot bind to protein A, whereas the bi-specific monovalent Fc diabodywill retain its ability to bind protein A via the protein A binding siteon the first polypeptide chain.

A preferred sequence for the CH2 and CH3 Domains of an antibody FcDomain present in the first polypeptide chain is (SEQ ID NO:56):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

A preferred sequence for the CH2 and CH3 Domains of an antibody FcDomain present in the third polypeptide chain is (SEQ ID NO:11):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHE ALHNRYTQKSLSLSPGK

C. DART-A w/Fc Version 1 Construct

In order to illustrate such Fc diabodies, the invention provides aDART-A w/Fc version 1 construct. The first polypeptide of the DART-Aw/Fc version 1 construct comprises, in the N-terminal to C-terminaldirection, an N-terminus, a VL domain of a monoclonal antibody capableof binding to CD123 (VL_(CD23)), an intervening linker peptide (Linker1), a VH domain of a monoclonal antibody capable of binding to CD3(VH_(CD33)), a Linker 2, an E-coil Domain, a Linker 5, Peptide 1, apolypeptide that contains the CH2 and CH3 Domains of an Fc Domain and aC-terminus. A preferred Linker 5 has the sequence (SEQ ID NO:32): GGG. Apreferred polypeptide that contains the CH2 and CH3 Domains of an FcDomain has the sequence (SEQ ID NO:37):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

Thus, the first polypeptide of such a DART-A w/Fc version 1 construct iscomposed of: SEQ ID NO:25—SEQ ID NO:29—SEQ ID NO:22—SEQ ID NO:30—SEQ IDNO:34—SEQ ID NO:32—SEQ ID NO:55—SEQ ID NO:37.

A preferred sequence of the first polypeptide of such a DART-A w/Fcversion 1 construct has the sequence (SEQ ID NO:13):

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGGGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK

A preferred polynucleotide encoding such a polypeptide is (SEQ IDNO:14):

gacttcgtgatgacacagtctcctgatagtctggccgtgagtctgggggagcgggtgactatgtcttgcaagagctcccagtcactgctgaacagcggaaatcagaaaaactatctgacctggtaccagcagaagccaggccagccccctaaactgctgatctattgggcttccaccagggaatctggcgtgcccgacagattcagcggcagcggcagcggcacagattttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactattgtcagaatgattacagctatccctacactttcggccaggggaccaagctggaaattaaaggaggcggatccggcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtccagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagcacatacgctatgaattgggtccgccaggctccagggaaggggctggagtgggttggaaggatcaggtccaagtacaacaattatgcaacctactatgccgactctgtgaaggatagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaattcttacgtgtcttggtttgcttattggggacaggggacactggtgactgtgtcttccggaggatgtggcggtggagaagtggccgcactggagaaagaggttgctgctttggagaaggaggtcgctgcacttgaaaaggaggtcgcagccctggagaaaggcggcggggacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa

The second chain of such a DART-A w/Fc version 1 construct willcomprise, in the N-terminal to C-terminal direction, an N-terminus, a VLdomain of a monoclonal antibody capable of binding to CD3 (VL_(CD3)), anintervening linker peptide (Linker 1), a VH domain of a monoclonalantibody capable of binding to CD123 (VH_(CD123)), a Linker 2, a K-coilDomain, and a C-terminus. Thus, the second polypeptide of such a DART-Aw/Fc version 1 construct is composed of: SEQ ID NO:21—SEQ ID NO:29—SEQID NO:26—SEQ ID NO:30—SEQ ID NO:35. Such a polypeptide has the sequence(SEQ ID NO:15):

QAVVTQEPSLTVSPGGIVTLICRSSTGAVITSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGEVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

A preferred polynucleotide encoding such a polypeptide has the sequence(SEQ ID NO:16):

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgaccctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattgggtgcagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaaagggctccctggacccctgcacggttttctggaagtctgctgggcggaaaggccgctctgactattaccggggcacaggccgaggacgaagccgattactattgtgctctgtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctgggagggggtggatccggcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtgaaggtgtcttgcaaagccagtggctacaccttcacagactactatatgaagtgggtcaggcaggctccaggacagggactggaatggatcggcgatatcattccttccaacggggccactttctacaatcagaagtttaaaggcagggtgactattaccgtggacaaatcaacaagcactgcttatatggagctgagctccctgcgctctgaagatacagccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattggggacagggcaccctggtgacagtgtcttccggaggatgtggcggtggaaaagtggccgcactgaaggagaaagttgctgctttgaaagagaaggtcgccgcacttaaggaaaaggt cgcagccctgaaagag

The third polypeptide chain of such a DART-A w/Fc version 1 willcomprise the CH2 and CH3 Domains of an IgG Fc Domain. A preferredpolypeptide that is composed of Peptide 1 (SEQ ID NO:55) and the CH2 andCH3 Domains of an Fc Domain (SEQ ID NO:11) and has the sequence of SEQID NO:54:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK

A preferred polynucleotide that encodes such a polypeptide has thesequence (SEQ ID NO:12):

gacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgagttgcgcagtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctcgtcagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccgctacacgcagaagagcctctccctgtctccgggtaaa

D. DART-A w/Fc Version 2 Construct

As a second example of such a DART-A w/Fc diabody, the inventionprovides a three chain diabody, “DART-A w/Fc Diabody Version 2” (FIG.3B).

The first polypeptide of such a DART-A w/Fc version 2 constructcomprises, in the N-terminal to C-terminal direction, an N-terminus, apeptide linker (Peptide 1), a polypeptide that contains the CH2 and CH3Domains of an Fc Domain linked (via a Linker 4) to the VL domain of amonoclonal antibody capable of binding to CD123 (VL_(CD23)), anintervening linker peptide (Linker 1), a VH domain of a monoclonalantibody capable of binding to CD3 (VH033), a Linker 2, a K-coil Domain,and a C-terminus.

A preferred polypeptide that contains the CH2 and CH3 Domains of an FcDomain has the sequence (SEQ ID NO:37):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

“Linker 4” will preferably comprise the amino acid sequence (SEQ IDNO:57): APSSS. A preferred “Linker 4” has the sequence (SEQ ID NO:33):APSSSPME. Thus, the first polypeptide of such a DART-A w/Fc version 2construct is composed of: SEQ ID NO:55—SEQ ID NO:37—SEQ ID NO:33—SEQ IDNO:25—SEQ ID NO:29—SEQ ID NO:22—SEQ ID NO:30—SEQ ID NO:35. A polypeptidehaving such a sequence is (SEQ ID NO:17):

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAPSSSPMEDFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGKVAALKEKVAALK EKVAALKEKVAALKE

A preferred polynucleotide encoding such a polypeptide has the sequence(SEQ ID NO:18):

gacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagccccttccagctcccctatggaagacttcgtgatgacacagtctcctgatagtctggccgtgagtctgggggagcgggtgactatgtcttgcaagagctcccagtcactgctgaacagcggaaatcagaaaaactatctgacctggtaccagcagaagccaggccagccccctaaactgctgatctattgggcttccaccagggaatctggcgtgcccgacagattcagcggcagcggcagcggcacagattttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactattgtcagaatgattacagctatccctacactttcggccaggggaccaagctggaaattaaaggaggcggatccggcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtccagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagcacatacgctatgaattgggtccgccaggctccagggaaggggctggagtgggttggaaggatcaggtccaagtacaacaattatgcaacctactatgccgactctgtgaaggatagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaattcttacgtgtcttggtttgcttattggggacaggggacactggtgactgtgtcttccggaggatgtggcggtggaaaagtggccgcactgaaggagaaagttgctgctttgaaagagaaggtcgccgcacttaaggaaaaggtcgcagccctgaaagag

The second polypeptide chain of such a DART-A w/Fc version 2 constructcomprises, in the N-terminal to C-terminal direction, the VL domain of amonoclonal antibody capable of binding to CD3 (VL_(CD3)), an interveninglinker peptide (Linker 1) and a VH domain of a monoclonal antibodycapable of binding to CD123 (VH_(CD23)). This portion of the molecule islinked (via Linker 2) to an E-coil Domain. Thus, the third polypeptideof such a DART-A w/Fc version 2 construct is composed of: SEQ IDNO:21—SEQ ID NO:29—SEQ ID NO:26—SEQ ID NO:30—SEQ ID NO:34. A polypeptidehaving such a sequence is (SEQ ID NO:1), and is preferably encoded by apolynucleotide having the sequence of SEQ ID NO:2.

The third polypeptide chain will comprise the CH2 and CH3 Domains of anIgG Fc Domain. A preferred polypeptide is composed of Peptide 1 (SEQ IDNO:55) and the CH2 and CH3 Domains of an Fc Domain (SEQ ID NO:11) andhas the sequence of SEQ ID NO:54.

In order to assess the activity of the above-mentioned CD123×CD3bi-specific diabodies (DART-A, DART-A w/ABD, DART-A w/Fc, DART-B), acontrol bi-specific diabody (Control DART) was produced. The ControlDART is capable of simultaneously binding to FITC and CD3. Its twopolypeptide chains have the following respective sequences:

Control DART Chain 1 (SEQ ID NO: 19):DVVMTQTPFSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEK Control DART Chain 2 (SEQ ID NO: 20):QAVVTQEPSLTVSPGGIVTLICRSSTGAVITSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGEVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSGGCGGGKVAALKEK VAALKEKVAALKEKVAALKE

IV. Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of the sequence-optimized CD123×CD3 bi-specific diabodies of thepresent invention, or a combination of such agents and apharmaceutically acceptable carrier. Preferably, compositions of theinvention comprise a prophylactically or therapeutically effectiveamount of the sequence-optimized CD123×CD3 bi-specific diabody of theinvention and a pharmaceutically acceptable carrier.

The invention also encompasses pharmaceutical compositions comprisingsequence-optimized CD123×CD3 bi-specific diabodies of the invention, anda second therapeutic antibody (e.g., tumor specific monoclonal antibody)that is specific for a particular cancer antigen, and a pharmaceuticallyacceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limited tothose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with sequence-optimized CD123×CD3 bi-specificdiabodies of the invention alone or with such pharmaceuticallyacceptable carrier. Additionally, one or more other prophylactic ortherapeutic agents useful for the treatment of a disease can also beincluded in the pharmaceutical pack or kit. The invention also providesa pharmaceutical pack or kit comprising one or more containers filledwith one or more of the ingredients of the pharmaceutical compositionsof the invention. Optionally associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

The present invention provides kits that can be used in the abovemethods. A kit can comprise sequence-optimized CD123×CD3 bi-specificdiabodies of the invention. The kit can further comprise one or moreother prophylactic and/or therapeutic agents useful for the treatment ofcancer, in one or more containers; and/or the kit can further compriseone or more cytotoxic antibodies that bind one or more cancer antigensassociated with cancer. In certain embodiments, the other prophylacticor therapeutic agent is a chemotherapeutic. In other embodiments, theprophylactic or therapeutic agent is a biological or hormonaltherapeutic.

V. Methods of Administration

The compositions of the present invention may be provided for thetreatment, prophylaxis, and amelioration of one or more symptomsassociated with a disease, disorder or infection by administering to asubject an effective amount of a fusion protein or a conjugated moleculeof the invention, or a pharmaceutical composition comprising a fusionprotein or a conjugated molecule of the invention. In a preferredaspect, such compositions are substantially purified (i.e.,substantially free from substances that limit its effect or produceundesired side effects). In a specific embodiment, the subject is ananimal, preferably a mammal such as non-primate (e.g., bovine, equine,feline, canine, rodent, etc.) or a primate (e.g., monkey such as, acynomolgus monkey, human, etc.). In a preferred embodiment, the subjectis a human.

Various delivery systems are known and can be used to administer thecompositions of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe antibody or fusion protein, receptor-mediated endocytosis (See,e.g., Wu et al. (1987) “Receptor-Mediated In Vitro Gene TransformationBy A Soluble DNA Carrier System,” J. Biol. Chem. 262:4429-4432),construction of a nucleic acid as part of a retroviral or other vector,etc.

Methods of administering a molecule of the invention include, but arenot limited to, parenteral administration (e.g., intradermal,intramuscular, intraperitoneal, intravenous and subcutaneous), epidural,and mucosal (e.g., intranasal and oral routes). In a specificembodiment, the sequence-optimized CD123×CD3 bi-specific diabodies ofthe invention are administered intramuscularly, intravenously, orsubcutaneously. The compositions may be administered by any convenientroute, for example, by infusion or bolus injection, by absorptionthrough epithelial or mucocutaneous linings (e.g., oral mucosa, rectaland intestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, pulmonary administration can also be employed, e.g., by use ofan inhaler or nebulizer, and formulation with an aerosolizing agent.See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272;5,874,064; 5,855,913; 5,290,540; and 4,800,078; and PCT Publication Nos.WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903,each of which is incorporated herein by reference in its entirety.

The invention also provides that the sequence-optimized CD123×CD3bi-specific diabodies of the invention are packaged in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof the molecule. In one embodiment, the sequence-optimized CD123×CD3bi-specific diabodies of the invention are supplied as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted, e.g., with water or saline to theappropriate concentration for administration to a subject. Preferably,the sequence-optimized CD123×CD3 bi-specific diabodies of the inventionare supplied as a dry sterile lyophilized powder in a hermeticallysealed container at a unit dosage of at least 5 μg, more preferably atleast 10 μg, at least 15 μg, at least 25 μg, at least 50 μg, at least100 μg, or at least 200 μg.

The lyophilized sequence-optimized CD123×CD3 bi-specific diabodies ofthe invention should be stored at between 2 and 8° C. in their originalcontainer and the molecules should be administered within 12 hours,preferably within 6 hours, within 5 hours, within 3 hours, or within 1hour after being reconstituted. In an alternative embodiment,sequence-optimized CD123×CD3 bi-specific diabodies of the invention aresupplied in liquid form in a hermetically sealed container indicatingthe quantity and concentration of the molecule, fusion protein, orconjugated molecule. Preferably, the liquid form of thesequence-optimized CD123×CD3 bi-specific diabodies of the invention aresupplied in a hermetically sealed container in which the molecules arepresent at a concentration of least 1 μg/ml, more preferably at least2.5 μg/ml, at least 5 μg/ml, at least 10 μg/ml, at least 50 μg/ml, or atleast 100 μg/ml.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For sequence-optimized CD123×CD3 bi-specific diabodies encompassed bythe invention, the dosage administered to a patient is preferablydetermined based upon the body weight (kg) of the recipient subject. Thedosage administered is typically from at least about 0.3 ng/kg per dayto about 0.9 ng/kg per day, from at least about 1 ng/kg per day to about3 ng/kg per day, from at least about 3 ng/kg per day to about 9 ng/kgper day, from at least about 10 ng/kg per day to about 30 ng/kg per day,from at least about 30 ng/kg per day to about 90 ng/kg per day, from atleast about 100 ng/kg per day to about 300 ng/kg per day, from at leastabout 200 ng/kg per day to about 600 ng/kg per day, from at least about300 ng/kg per day to about 900 ng/kg per day, from at least about 400ng/kg per day to about 800 ng/kg per day, from at least about 500 ng/kgper day to about 1000 ng/kg per day, from at least about 600 ng/kg perday to about 1000 ng/kg per day, from at least about 700 ng/kg per dayto about 1000 ng/kg per day, from at least about 800 ng/kg per day toabout 1000 ng/kg per day, from at least about 900 ng/kg per day to about1000 ng/kg per day, or at least about 1,000 ng/kg per day.

In another embodiment, the patient is administered a treatment regimencomprising one or more doses of such prophylactically or therapeuticallyeffective amount of the sequence-optimized CD123×CD3 bi-specificdiabodies encompassed by the invention, wherein the treatment regimen isadministered over 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. Incertain embodiments, the treatment regimen comprises intermittentlyadministering doses of the prophylactically or therapeutically effectiveamount of the sequence-optimized CD123×CD3 bi-specific diabodiesencompassed by the invention (for example, administering a dose on day1, day 2, day 3 and day 4 of a given week and not administering doses ofthe prophylactically or therapeutically effective amount of thesequence-optimized CD123×CD3 bi-specific diabodies encompassed by theinvention on day 5, day 6 and day 7 of the same week). Typically, thereare 1, 2, 3, 4, 5 or more courses of treatment. Each course may be thesame regimen or a different regimen.

In another embodiment, the administered dose escalates over the firstquarter, first half or first two-thirds or three-quarters of theregimen(s) (e.g., over the first, second, or third regimens of a 4course treatment) until the daily prophylactically or therapeuticallyeffective amount of the sequence-optimized CD123×CD3 bi-specificdiabodies encompassed by the invention is achieved.

Table 1 provides 5 examples of different dosing regimens described abovefor a typical course of treatment.

TABLE 1 Regimen Day Diabody Dosage (ng diabody per kg subject weight perday) 1 1, 2, 3, 4 100 100 100 100 100 5, 6, 7 none none none none none 21, 2, 3, 4 300 500 700 900 1,000 5, 6, 7 none none none none none 3 1,2, 3, 4 300 500 700 900 1,000 5, 6, 7 none none none none none 4 1, 2,3, 4 300 500 700 900 1,000 5, 6, 7 none none none none none

The dosage and frequency of administration of sequence-optimizedCD123×CD3 bi-specific diabodies of the invention may be reduced oraltered by enhancing uptake and tissue penetration of thesequence-optimized CD123×CD3 bi-specific diabodies by modifications suchas, for example, lipidation.

The dosage of the sequence-optimized CD123×CD3 bi-specific diabodies ofthe invention administered to a patient may be calculated for use as asingle agent therapy. Alternatively, the sequence-optimized CD123×CD3bi-specific diabodies of the invention are used in combination withother therapeutic compositions and the dosage administered to a patientare lower than when said molecules are used as a single agent therapy.

The pharmaceutical compositions of the invention may be administeredlocally to the area in need of treatment; this may be achieved by, forexample, and not by way of limitation, local infusion, by injection, orby means of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a molecule of the invention,care must be taken to use materials to which the molecule does notabsorb.

The compositions of the invention can be delivered in a vesicle, inparticular a liposome (See Langer (1990) “New Methods Of Drug Delivery,”Science 249:1527-1533); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 3 17-327; seegenerally ibid.).

The compositions of the invention can be delivered in acontrolled-release or sustained-release system. Any technique known toone of skill in the art can be used to produce sustained-releaseformulations comprising one or more sequence-optimized CD123×CD3bi-specific diabodies of the invention. See, e.g., U.S. Pat. No.4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698;Ning et al. (1996) “Intratumoral Radioimmunotheraphy Of A Human ColonCancer Xenograft Using A Sustained-Release Gel,” Radiotherapy & Oncology39:179-189, Song et al. (1995) “Antibody Mediated Lung Targeting OfLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397; Cleek et al. (1997) “Biodegradable PolymericCarriers For A bFGF Antibody For Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al.(1997) “Microencapsulation Of Recombinant Humanized Monoclonal AntibodyFor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in itsentirety. In one embodiment, a pump may be used in a controlled-releasesystem (See Langer, supra; Sefton, (1987) “Implantable Pumps,” CRC Crit.Rev. Biomed. Eng. 14:201-240; Buchwald et al. (1980) “Long-Term,Continuous Intravenous Heparin Administration By An Implantable InfusionPump In Ambulatory Patients With Recurrent Venous Thrombosis,” Surgery88:507-516; and Saudek et al. (1989) “A Preliminary Trial Of TheProgrammable Implantable Medication System For Insulin Delivery,” N.Engl. J. Med. 321:574-579). In another embodiment, polymeric materialscan be used to achieve controlled-release of the molecules (see e.g.,MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974); CONTROLLED DRUG BIOAVAILABILITY, DRUGPRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York(1984); Levy et al. (1985) “Inhibition Of Calcification Of BioprostheticHeart Valves By Local Controlled-Release Diphosphonate,” Science228:190-192; During et al. (1989) “Controlled Release Of Dopamine From APolymeric Brain Implant: In Vivo Characterization,” Ann. Neurol.25:351-356; Howard et al. (1989) “Intracerebral Drug Delivery In RatsWith Lesion Induced Memory Deficits,” J. Neurosurg. 7(1):105-112); U.S.Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCTPublication No. WO 99/15154; and PCT Publication No. WO 99/20253).Examples of polymers used in sustained-release formulations include, butare not limited to, poly(-hydroxy ethyl methacrylate), poly(methylmethacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. A controlled-release system can be placedin proximity of the therapeutic target (e.g., the lungs), thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MEDICALAPPLICATIONS OF CONTROLLED RELEASE, supra, vol. 2, pp. 115-138 (1984)).Polymeric compositions useful as controlled-release implants can be usedaccording to Dunn et al. (See U.S. Pat. No. 5,945,115). This particularmethod is based upon the therapeutic effect of the in situcontrolled-release of the bioactive material from the polymer system.The implantation can generally occur anywhere within the body of thepatient in need of therapeutic treatment. A non-polymeric sustaineddelivery system can be used, whereby a non-polymeric implant in the bodyof the subject is used as a drug delivery system. Upon implantation inthe body, the organic solvent of the implant will dissipate, disperse,or leach from the composition into surrounding tissue fluid, and thenon-polymeric material will gradually coagulate or precipitate to form asolid, microporous matrix (See U.S. Pat. No. 5,888,533).

Controlled-release systems are discussed in the review by Langer (1990,“New Methods Of Drug Delivery,” Science 249:1527-1533). Any techniqueknown to one of skill in the art can be used to producesustained-release formulations comprising one or more therapeutic agentsof the invention. See, e.g., U.S. Pat. No. 4,526,938; InternationalPublication Nos. WO 91/05548 and WO 96/20698; Ning et al. (1996)“Intratumoral Radioimmunotheraphy Of A Human Colon Cancer XenograftUsing A Sustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Songet al. (1995) “Antibody Mediated Lung Targeting Of Long-CirculatingEmulsions,” PDA Journal of Pharmaceutical Science & Technology50:372-397; Cleek et al. (1997) “Biodegradable Polymeric Carriers For AbFGF Antibody For Cardiovascular Application,” Pro. Int'l. Symp.Control. Rel. Bioact. Mater. 24:853-854; and Lam et al. (1997)“Microencapsulation Of Recombinant Humanized Monoclonal Antibody ForLocal Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in itsentirety.

Where the composition of the invention is a nucleic acid encoding asequence-optimized CD123×CD3 bi-specific diabody of the invention, thenucleic acid can be administered in vivo to promote expression of itsencoded sequence-optimized CD123×CD3 bi-specific diabody, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al. (1991) “Antennapedia Homeobox Peptide Regulates NeuralMorphogenesis,” Proc. Natl. Acad. Sci. (U.S.A.) 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of sequence-optimized CD123×CD3 bi-specific diabodiesof the invention can include a single treatment or, preferably, caninclude a series of treatments. In a preferred example, a subject istreated with sequence-optimized CD123×CD3 bi-specific diabodies of theinvention one time per week for between about 1 to 10 weeks, preferablybetween 2 to 8 weeks, more preferably between about 3 to 7 weeks, andeven more preferably for about 4, 5, or 6 weeks. The pharmaceuticalcompositions of the invention can be administered once a day, twice aday, or three times a day. Alternatively, the pharmaceuticalcompositions can be administered once a week, twice a week, once everytwo weeks, once a month, once every six weeks, once every two months,twice a year or once per year. It will also be appreciated that theeffective dosage of the molecules used for treatment may increase ordecrease over the course of a particular treatment.

VI. Uses of the Compositions of the Invention

The sequence-optimized CD123×CD3 bi-specific diabodies of the presentinvention have the ability to treat any disease or condition associatedwith or characterized by the expression of CD123. Thus, withoutlimitation, such molecules may be employed in the diagnosis or treatmentof acute myeloid leukemia (AML), chronic myelogenous leukemia (CML),including blastic crisis of CML and Abelson oncogene associated with CML(Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute Blymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL),including Richter's syndrome or Richter's transformation of CLL, hairycell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm(BPDCN), non-Hodgkin lymphomas (NHL), including mantel cell leukemia(MCL), and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma,systemic mastocytosis, and Burkitt's lymphoma (see Example 2);Autoimmune Lupus (SLE), allergy, asthma and rheumatoid arthritis. Thebi-specific diabodies of the present invention may additionally be usedin the manufacture of medicaments for the treatment of theabove-described conditions.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention unless specified.

Example 1 Construction of CD123×CD3 Bi-Specific Diabodies and ControlProtein

Table 2 contains a list of bi-specific diabodies that were expressed andpurified. Sequence-optimized CD123×CD3 bi-specific diabody (DART-A) andnon-sequence-optimized CD123×CD3 bi-specific diabody (DART-B) arecapable of simultaneously binding to CD123 and CD3. The controlbi-specific diabody (Control DART) is capable of simultaneously bindingto FITC and CD3. The bi-specific diabodies are heterodimers orheterotrimers of the recited amino acid sequences. Methods for formingbi-specific diabodies are provided in WO 2006/113665, WO 2008/157379, WO2010/080538, WO 2012/018687, WO 2012/162068 and WO 2012/162067.

TABLE 2 Polypeptide Chain Amino Acid Nucleic Acid Bi-Specific DiabodiesSequences Encoding Sequences Sequence-Optimized CD123 × CD3 SEQ ID NO: 1SEQ ID NO: 2 Bi-Specific Diabody (DART-A) SEQ ID NO: 3 SEQ ID NO: 4(Binds to CD3 at epitope 1) Non-Sequence-Optimized CD123 × SEQ ID NO: 5SEQ ID NO: 6 CD3 Bi-Specific Diabody (DART-B) SEQ ID NO: 7 SEQ ID NO: 8(Binds to CD3 at epitope 2) Sequence-Optimized CD123 × CD3 SEQ ID NO: 9SEQ ID NO: 10 Bi-Specific Diabody Having an SEQ ID NO: 3 SEQ ID NO: 4Albumin-Binding Domain (DART-A w/ABD) (Binds to CD3 at epitope 1)Comprises an Albumin-Binding Domain (ABD) for extension of half- life invivo Sequence-Optimized CD123 × CD3 SEQ ID NO: 54 SEQ ID NO: 12Bi-Specific Diabody Having an IgG SEQ ID NO: 13 SEQ ID NO: 14 Fc DomainVersion 1 (DART-A w/Fc SEQ ID NO: 15 SEQ ID NO: 16 version 1) (Binds toCD3 at epitope 1) Comprises an Fc Domain for extension of half-life invivo Sequence-Optimized CD123 × CD3 SEQ ID NO: 54 SEQ ID NO: 12Bi-Specific Diabody Having an IgG SEQ ID NO: 17 SEQ ID NO: 18 Fc DomainVersion 2 (DART-A w/Fc SEQ ID NO: 1 SEQ ID NO: 2 version 2) (Binds toCD3 at epitope 1) Comprises an Fc Domain for extension of half-life invivo Control Bi-Specific Diabody (Control SEQ ID NO: 19 DART (or ControlDART) SEQ ID NO: 20 (Binds to CD3 at epitope 1) (Binds to an irrelevanttarget - FITC)

Example 2 Antibody Labeling of Target Cells for Quantitative FACS(QFACS)

A total of 10⁶ target cells were harvested from the culture, resuspendedin 10% human AB serum in FACS buffer (PBS+1% BSA+0.1% NaAzide) andincubated for 5 min for blocking Fc receptors. Antibody labeling ofmicrospheres with different antibody binding capacities (Quantum™ SimplyCellular® (QSC), Bangs Laboratories, Inc., Fishers, Ind.) and targetcells were labeled with anti-CD123 PE antibody (BD Biosciences)according to the manufacturer's instructions. Briefly, one drop of eachQSC microsphere was added to a 5 mL polypropylene tube and PElabeled-anti-CD123 antibody was added at 1 μg/mL concentration to bothtarget cells and microspheres. Tubes were incubated in the dark for 30minutes at 4° C. Cells and microspheres were washed by adding 2 mL FACSbuffer and centrifuging at 2500×G for 5 minutes. One drop of the blankmicrosphere population was added after washing. Microspheres wereanalyzed first on the flow cytometer to set the test-specific instrumentsettings (PMT voltages and compensation). Using the same instrumentsettings, geometric mean fluorescence values of microspheres and targetcells were recorded. A standard curve of antibody binding sites onmicrosphere populations was generated from geometric mean fluorescenceof microsphere populations. Antibody binding sites on target cells werecalculated based on geometric mean fluorescence of target cells usingthe standard curve generated for microspheres in QuickCal spreadsheet(Bangs Laboratories).

To determine suitable target cell lines for evaluating CD123×CD3bi-specific diabodies, CD123 surface expression levels on target linesKasumi-3 (AML), Molm13 (AML), THP-1 (AML), TF-1 (Erythroleukemia), andRS4-11 (ALL) were evaluated by quantitative FACS (QFACS). Absolutenumbers of CD123 antibody binding sites on the cell surface werecalculated using a QFACS kit. As shown in Table 3, the absolute numberof CD123 antibody binding sites on cell lines were in the order ofKasumi-3 (high)>Molm13 (medium)>THP-1(medium)>TF-1 (medium low)>RS4-11(low). The three highest expressing cell lines were the AML cell lines:Kasumi-3, MOLM13, and THP-1. The non-AML cell lines: TF-1 and RS4-11 hadmedium-low/low expression of CD123, respectively.

TABLE 3 Target Cell CD123 Surface Expression Line (Antibody BindingSites) Kasumi-3 118620 Molm13 27311 THP-1 58316 TF-1 14163 RS4-11 957A498 Negative HT29 Negative

Example 3 CTL Cytotoxicity Assay (LDH Release Assay)

Adherent target tumor cells were detached with 0.25% Trypsin-EDTAsolution and collected by centrifugation at 1000 rpm for 5 min.Suspension target cell lines were harvested from the culture, washedwith assay medium. The cell concentration and viability were measured byTrypan Blue exclusion using a Beckman Coulter Vi-Cell counter. Thetarget cells were diluted to 4×10⁵ cells/mL in the assay medium. 50 μLof the diluted cell suspension was added to a 96-well U-bottom cellculture treated plate (BD Falcon Cat #353077).

Three sets of controls to measure target maximal release (MR), antibodyindependent cellular cytotoxicity (AICC) and target cell spontaneousrelease (SR) were set up as follows:

-   -   1) MR: 200 μL assay medium without CD123×CD3 bi-specific        diabodies and 50 μL target cells; detergent added at the end of        the experiment to determine the maximal LDH release.    -   2) AICC: 50 μL assay medium without CD123×CD3 bi-specific        diabodies, 50 μL, target cells and 100 μL, T cells.    -   3) SR: 150 μL, medium without CD123×CD3 bi-specific diabodies        and 50 μL, target cells.

CD123×CD3 bi-specific diabodies (DART-A, DART-A w/ABD and DART-B) andcontrols were initially diluted to a concentration of 4 μg/mL, andserial dilutions were then prepared down to a final concentration of0.00004 ng/mL (i.e., 40 fg/mL). 50 μL, of dilutions were added to theplate containing 50 μL target cells/well.

Purified T cells were washed once with assay medium and resuspended inassay medium at cell density of 2×10⁶ cells/mL. 2×10⁵ T cells in 100 μL,were added to each well, for a final effector-to-target cell (E:T) ratioof 10:1. Plates were incubated for approximately 18 hr at 37° C. in 5%CO₂.

Following incubation, 25 μL of 10× lysis solution (Promega #G182A) or 1mg/mL digitonin was added to the maximum release control wells, mixed bypipetting 3 times and plates were incubated for 10 min to completelylyse the target cells. The plates were centrifuged at 1200 rpm for 5minutes and 50 μL of supernatant were transferred from each assay platewell to a flat bottom ELISA plate and 50 μl of LDH substrate solution(Promega #G1780) was added to each well. Plates were incubated for 10-20min at room temperature (RT) in the dark, then 50 μL, of Stop solutionwas added. The optical density (O.D.) was measured at 490 nm within 1hour on a Victor2 Multilabel plate reader (Perkin Elmer #1420-014). Thepercent cytotoxicity was calculated as described below and dose-responsecurves were generated using GraphPad PRISMS® software.

Specific cell lysis was calculated from O.D. data using the followingformula:

Cytotoxicity (%)=100×(OD of Sample−OD of AICC)/(OD of MR−OD of SR)

Redirected Killing of Target Cell Lines with Different Levels of CD123Surface Levels:

The CD123×CD3 bi-specific diabodies exhibited a potent redirectedkilling ability with concentrations required to achieve 50% of maximalactivity (EC50s) in sub-ng/mL range, regardless of CD3 epitope bindingspecificity (DART-A versus DART-B) in target cell lines with high CD123expression, Kasumi-3 (EC50=0.01 ng/mL) (FIG. 4 Panel D), mediumCD123-expression, Molm13 (EC50=0.18 ng/mL) and THP-1 (EC50=0.24 ng/mL)(FIG. 4, Panel C and E, respectively) and medium low or low CD123expression, TF-1 (EC50=0.46 ng/mL) and RS4-11 (EC50=0.5 ng/mL) (FIG. 4,Panel B and A, respectively). Similarly, CD123×CD3 bi-specific moleculesmediated redirected killing was also observed with multiple target celllines with T cells from different donors and no redirected killingactivity was observed in cell lines that do not express CD123. Resultsare summarized in Table 4.

TABLE 4 EC50 of Sequence- CD123 surface optimized CD123 × expression CD3bi-specific (antibody binding diabodies (ng/mL) Target cell line sites)E:T = 10:1 Max % killing Kasumi-3 118620 0.01 94 Molm13 27311 0.18 43THP-1 58316 0.24 40 TF-1 14163 0.46 46 RS4-11 957 0.5 60 A498 NegativeNo activity No activity HT29 Negative No activity No activity

Should it be necessary to replicate this example it will be appreciatedthat one of skill in the art may, within reasonable and acceptablelimits, vary the above-described protocol in a manner appropriate forreplicating the described results. Thus, the exemplified protocol is notintended to be adhered to in a precisely rigid manner.

Example 4 T Cell Activation During Redirected Killing bySequence-Optimized CD123×CD3 Bi-Specific Diabodies (DART-A, DART-A w/ABDand DART-A w/Fc)

The sequence-optimized CD123×CD3 bi-specific diabodies exhibited apotent redirected killing ability regardless of the presence or absenceof half-life extension technology (DART-A versus DART-A w/ABD versusDART-A w/Fc) in target cell lines with high CD123 expression, Kasumi-3,and medium, THP-1, CD123-expression, (FIG. 5, Panels A and B,respectively) To characterize T cell activation duringsequence-optimized CD123×CD3 bi-specific diabody mediated redirectedkilling process, T cells from redirected killing assays were stained forT cell activation marker CD25 and analyzed by FACS. As shown in FIG. 5,Panel D, CD25 was up-regulated in CD8 T cells in a dose-dependent mannerindicating that sequence-optimized CD123×CD3 bi-specific diabodiesinduce T cell activation in the process of redirected killing.Conversely, in the absence of target cells there is no activation of CD8T cells (FIG. 5, Panel C) indicating the sequence-optimized CD123×CD3bi-specific diabodies do not activate T cells in the absence of targetcells. Likewise, CD8 T cells are not activated when incubated withtarget cells and a control bi-specific diabody (Control DART) (FIG. 5,Panel D) indicating the requirement of cross-linking the T cell andtarget cell with the sequence-optimized CD123×CD3 bi-specific diabodies.

Example 5 Intracellular Staining for Granzyme B and Perforin

To determine the intracellular levels of granzyme B and perforin in Tcells, a CTL assay was setup as described above. After approximately 18h, cells from the assay plate were stained with anti-CD4 and anti-CD8antibodies by incubating for 30 minutes at 4° C. Following surfacestaining, cells were incubated in 100 μL fixation and permeabilizationbuffer (BD BioSciences) for 20 min at 4° C. Cells were washed withpermeabilization/wash buffer (BD BioSciences) and incubated in 50 μL ofgranzyme B and a perforin antibody mixture (prepared in 1×permeabilization/wash buffer) at 4° C. for 30 minutes. Then cells werewashed with 250 μL permeabilization/wash buffer and resuspended inpermeabilization/wash buffer for FACS acquisition.

Upregulation of Granzyme B and Perforin by Sequence-Optimized CD123×CD3Bi-Specific Diabody (DART-A) in T Cells During Redirected Killing

To investigate the possible mechanism for sequence-optimized CD123×CD3bi-specific diabody (DART-A) mediated cytotoxicity by T cells,intracellular granzyme B and perforin levels were measured in T cellsafter the redirected killing. Dose-dependent upregulation of granzyme Band perforin levels in both CD8 and CD4 T cells was observed followingincubation of T cells and Kasumi-3 cells with DART-A (FIG. 6, Panel A).Interestingly, the upregulation was almost two-fold higher in CD8 Tcells compared to CD4 T cells (FIG. 6, Panel A). When the assay wasperformed in the presence of granzyme B and perforin inhibitors no cellkilling was observed. There was no upregulation of granzyme B orperforin in CD8 or CD4 T cells when T cells were incubated with Kasumi-3target cells and a control bi-specific diabody (Control DART) (FIG. 5,Panel B). These data indicate that DART-A mediated target cell killingmay be mediated through granzyme B and perforin mechanisms.

Example 6 In Vivo Antitumor Activity of Sequence-Optimized CD123×CD3Bi-Specific Diabody (DART-A)

Isolation of PBMCs and T Cells from Human Whole Blood

PBMCs from healthy human donors were isolated from whole blood by usingFicoll gradient centrifugation. In brief, whole blood was diluted 1:1with sterile PBS. Thirty-five mL of the diluted blood was layered onto15 mL Ficoll-Paque™ Plus in 50-mL tubes and the tubes were centrifugedat 1400 rpm for 20 min with the brake off The buffy coat layer betweenthe two phases was collected into a 50 mL tube and washed with 45 mL PBSby centrifuging the tubes at 600×g (1620 rpm) for 5 min. The supernatantwas discarded and the cell pellet was washed once with PBS and viablecell count was determined by Trypan Blue dye exclusion. The PBMCs wereresuspended to a final concentration of 2.5×10⁶ cells/mL in completemedium (RPMI 1640, 10% FBS, 2 mM Glutamine, 10 mM HEPES, 100μ/100μ/mLpenicillin/Streptomycin (P/S).

T Cell Isolation:

Untouched T cells were isolated by negative selection from PBMCs fromhuman whole blood using Dynabeads Untouched Human T Cell isolation kit(Life Technologies) according to manufacturer's instructions. After theisolation, T cells were cultured overnight in RPMI medium with 10% FBS,1% penicillin/Streptomycin.

Tumor Model

Human T cells and tumor cells (Molm13 or RS4-11) were combined at aratio of 1:5 (1×10⁶ and 5×10⁶, respectively) and suspended in 200 μL ofsterile saline and injected subcutaneously (SC) on Study Day 0 (SD0).Sequence-optimized CD123×CD3 bi-specific diabody (DART-A) or a controlbi-specific diabody (Control DART) were administered intravenously (IV)via tail vein injections in 100 μL as outlined in Table 5 (MOLM13) andTable 6 (RS4-11).

TABLE 5 Study Design for MOLM13 Model Treatment Dose Number of Group(mg/kg) Schedule Animals Vehicle Control — SD0, 1, 2, 3 8 (MOLM-13 cellsalone implanted or + T cells) DART-A 0.5 SD0, 1, 2, 3 8 DART-A 0.2 SD0,1, 2, 3 8 DART-A 0.1 SD0, 1, 2, 3 8 DART-A 0.02 SD0, 1, 2, 3 8 DART-A0.004 SD0, 1, 2, 3 8 DART-A 0.0008 SD0, 1, 2, 3 8 DART-A 0.00016 SD0, 1,2, 3 8

TABLE 6 Study Design for RS4-11 Model Treatment Dose Number of Group(mg/kg) Schedule Animals Vehicle Control — SD0, 1, 2, 3 8 (RS4-11 cellsalone implanted) Vehicle Control — SD0, 1, 2, 3 8 (RS4-11 + T cellsimplanted) Control DART 0.2 SD0, 1, 2, 3 8 DART-A 0.5 SD0, 1, 2, 3 8DART-A 0.2 SD0, 1, 2, 3 8 DART-A 0.1 SD0, 1, 2, 3 8 DART-A 0.02 SD0, 1,2, 3 8 DART-A 0.004 SD0, 1, 2, 3 8

Data Collection and Statistical Analysis:

Animal Weights—

Individual animal weights were recorded twice weekly until studycompletion beginning at the time of tumor cell injection.

Moribundity/Mortality—

Animals were observed twice weekly for general moribundity and daily formortality. Animal deaths were assessed as drug-related or technicalbased on factors including gross observation and weight loss; animaldeaths were recorded daily.

Tumor Volume—

Individual tumor volumes were recorded twice weekly beginning within oneweek of tumor implantation and continuing until study completion.

${{Tumor}\mspace{14mu} {Volume}\mspace{14mu} \left( {mm}^{3} \right)} = \frac{{Length}\mspace{14mu} ({mm}) \times {width}^{2}}{2}$

Animals experiencing technical or drug-related deaths were censored fromthe data calculations.

Tumor Growth Inhibition—

Tumor growth inhibition (TGI) values were calculated for each groupcontaining treated animals using the formula:

$1 - {\frac{\begin{matrix}{{{Mean}\mspace{14mu} {Final}\mspace{14mu} {Tumor}\mspace{14mu} {Volume}\mspace{14mu} ({Treated})} -} \\{{Mean}\mspace{14mu} {Initial}\mspace{14mu} {Tumor}\mspace{14mu} {Volume}\mspace{14mu} ({Treated})}\end{matrix}}{\begin{matrix}{{{Mean}\mspace{14mu} {Final}\mspace{14mu} {Tumor}\mspace{14mu} {Volume}\mspace{14mu} ({Control})} -} \\{{Mean}\mspace{14mu} {Initial}\mspace{14mu} {Tumor}\mspace{14mu} {Volume}\mspace{14mu} ({Control})}\end{matrix}} \times 100}$

Animals experiencing a partial or complete response, or animalsexperiencing technical or drug-related deaths were censored from the TGIcalculations. The National Cancer Institute criteria for compoundactivity is TGI>58% (Corbett et al. (2004) Anticancer Drug DevelopmentGuide; Totowa, N.J.: Humana 99-123).

Partial/Complete Tumor Response—

Individual mice possessing tumors measuring less than 1 mm³ on Day 1were classified as having partial response (PR) and a percent tumorregression (% TR) value was determined using the formula:

$1 - {\frac{{Final}\mspace{14mu} {Tumor}\mspace{14mu} {Volume}\mspace{14mu} \left( {mm}^{3} \right)}{{Initial}\mspace{14mu} {Tumor}\mspace{14mu} {Volume}\mspace{14mu} \left( {mm}^{3} \right)} \times 100\%}$

Individual mice lacking palpable tumors were classified as undergoing acomplete response (CR).

Tumor Volume Statistics—

Statistical analyses were carried out between treated and control groupscomparing tumor volumes. For these analyses, a two-way analyses ofvariance followed by a Bonferroni post-test were employed. All analyseswere performed using GraphPad PRISM® software (version 5.02). Weight andtumor data from individual animals experiencing technical ordrug-related deaths were censored from analysis. However, tumor datafrom animals reporting partial or complete responses were included inthese calculations.

MOLM13 Results

The AML cell line MOLM13 was pre-mixed with activated T cells andimplanted SC in NOD/SCID gamma (NSG) knockout mice (N=8/group) on SD0 asdetailed above. The MOLM13 tumors in the vehicle-treated group (MOLM13cells alone or plus T cells) demonstrated a relatively aggressive growthprofile in vivo (FIG. 7, Panels A and B). At SD8, the average volume ofthe tumors in the vehicle-treated group was 129.8±29.5 mm³ and by SD15the tumors had reached an average volume of 786.4±156.7 mm³. By the endof the experiment on SD18, the tumors had reached an average volume of1398.8±236.9 mm³.

Treatment with DART-A was initiated on the same day the tumor cell/Tcell mixture was implanted [(SD0)] and proceeded subsequently with dailyinjections for an additional 7 days for a total of 8 daily injections.The animals were treated with DART-A at 9 dose levels (0.5, 0.2, 0.1,0.02, and 0.004 mg/kg and 20, 4, 0.8 and 0.16 μg/kg). Results are shownin FIG. 7, Panel A (0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg) and FIG. 7,Panel B (20, 4, 0.8 and 0.16 μg/kg). By Study Day 11, the growth of theMOLM13 tumors was significantly inhibited at the 0.16, 0.5, 0.2, 0.1,0.02, and 0.004 mg/kg dose levels (p<0.001). Moreover, the treatment ofthe MOLM13 tumor-bearing mice at the 20 and 4 μg/kg dose levels resultedin 8/8 and 7/8 CRs, respectively. By the end of the experiment on SD18,the average volume of the tumors treated with DART-A at the 0.8-20μg/kg) ranged from 713.6.0±267.4 to 0 mm³, all of which weresignificantly smaller than the tumors in the vehicle-treated controlgroup. The TGI values were 100, 94, and 49% for the 20, 4, and 0.8 μg/kgdose groups, respectively. In comparison to the vehicle-treated MOLM13tumor cell group, the groups that received DART-A at the 20 and 4 μg/kgdose level reached statistical significance by SD15 while the grouptreated with 0.8 μg/kg reached significance on SD18.

RS4-11 Results

The ALL cell line RS4-11 was pre-mixed with activated T cells andimplanted SC in NOD/SCID gamma knockout mice (N=8/group) on SD0 asdetailed above. The RS4-11 tumors in the vehicle-treated group (RS4-11cells alone or plus T cells) demonstrated a relatively aggressive growthprofile in vivo (FIG. 8).

Treatment with DART-A was initiated on the same day the tumor cell/Tcell mixture was implanted [(SD0)] and proceeded subsequently with dailyinjections for an additional 3 days for a total of 4 daily injections.The animals were treated with DART-A at 5 dose levels (0.5, 0.2, 0.1,0.02, and 0.004 mg/kg). Results are shown in FIG. 8.

Sequence-optimized CD123×CD3 bi-specific diabody (DART-A) effectivelyinhibited the growth of both MOLM13 AML and RS4-11ALL tumors implantedSC in NOD/SCID mice in the context of the Winn model when dosing wasinitiated on the day of implantation and continued for 3 or moreconsecutive days. Based on the criteria established by the NationalCancer Institute, DART-A at the 0.1 mg/kg dose level and higher (TGI>58)is considered active in the RS4-11 model and an DART-A dose of 0.004mg/kg and higher was active in the MOLM13 model. The lower DART-A dosesassociated with the inhibition of tumor growth in the MOLM13 modelcompared with the RS4-11 model are consistent with the in vitro datademonstrating that MOLM13 cells have a higher level of CD123 expressionthan RS4-11 cells, which correlated with increased sensitivity to DART-Amediated cytotoxicity in vitro in MOLM13 cells.

Should it be necessary to replicate this example it will be appreciatedthat one of skill in the art may, within reasonable and acceptablelimits, vary the above-described protocol in a manner appropriate forreplicating the described results. Thus, the exemplified protocol is notintended to be adhered to in a precisely rigid manner.

Example 7 CD123 Surface Expression on Leukemic Blast Cells and StemCells in Primary Tissue Sample from AML Patient 1

To define the CD123 expression pattern in AML patient 1 primary samples,cryopreserved primary AML patient bone marrow and PBMC samples wereevaluated for CD123 surface expression on leukemic blast cells.

AML Bone Marrow Sample—Clinical Report

-   -   Age: 42    -   Gender: Female    -   AML Subtype: M2    -   Cancer cell percentage based on morphology: 67.5%    -   Bone marrow immunophenotyping:    -   CD15=19%, CD33=98.5%, CD38=28.8%, CD45=81.8%, CD64=39.7%,    -   CD117=42.9%, HLA-DR=17%, CD2=1.8%, CD5=0.53%, CD7=0.2%,    -   CD10=0.41%, CD19=1.1%, CD20=1.4%, CD22=0.71% CD34=0.82%

CD123 Expression in Leukemic Blast Cells in Bone Marrow Mononucleocytes(BM MNC)

A total of 0.5×10⁶ bone marrow mononucleocytes (BM MNC) and peripheralblood mononucleocytes (PBMC)) from AML patient 1 were evaluated forCD123 expression. The Kasumi-3 cell line was included as a control.Leukemic blast cells were identified using the myeloid marker CD33. Asshown in FIG. 9, Panel A, 87% of the cells from AML bone marrow frompatient 1 expressed CD123 and CD33. CD123 expression levels wereslightly lower than the CD123 high-expressing Kasumi-3 AML cell line(FIG. 9, Panel B).

Example 8 Autologous CTL Killing Assay Using AML Patient PrimarySpecimens

Cryopreserved primary AML specimen (bone marrow mononucleocytes (BMNC)and peripheral blood mononucleocytes (PBMC)) from AML patient 1 werethawed in RPMI 1640 with 10% FBS and allowed to recover overnight at 37°C. in 5% CO₂. Cells were washed with assay medium (RPMI 1640+10% FBS)and viable cell count was determined by Trypan Blue exclusion. 150,000cells/well in 150 μL assay medium were added to 96-well U-bottom plate(BD Biosciences). Sequence-optimized CD123×CD3 bi-specific diabody(DART-A) was diluted to 0.1, and 0.01 ng/mL and 50 μL of each dilutionwas added to each well (final volume=200 μL). Control bi-specificdiabody (Control DART) was diluted to 0.1 ng/mL and 50 μL of eachdilution was added to each well (final volume=200 μL). A separate assayplate was set up for each time point (48, 72, 120 and 144 hours) andplates were incubated at 37° C. in a 5% CO2 incubator. At each timepoint, cells were stained with CD4, CD8, CD25, CD45, CD33, and CD123antibodies. Labeled cells were analyzed in FACS Calibur flow cytometerequipped with CellQuest Pro acquisition software, Version 5.2.1 (BDBiosciences). Data analysis was performed using Flowjo v9.3.3 software(Treestar, Inc). T cell expansion was measured by gating on CD4+ andCD8+ populations and activation was determined by measuring CD25 meanfluorescent intensity (MFI) on the CD4+ and CD8+-gated populations.Leukemic blast cell population was identified by CD45+CD33+ gating.

Autologous Tumor Cell Depletion, T Cell Expansion and Activation bySequence-Optimized CD123×CD3 Bi-Specific Diabody (DART-A) in PrimarySpecimens from AML Patient 1

To determine the sequence-optimized CD123×CD3 bi-specific diabody(DART-A) mediated activity in AML patient 1, patient samples wereincubated with 0.1 ng/mL or 0.01 ng/mL of DART-A and percentages ofleukemic blast cells and T cells were measured at different time pointsfollowing the treatment. Leukemic blast cells were identified byCD45+/CD33+ gating. Incubation of primary AML bone marrow samples withDART-A resulted in depletion of the leukemic cell population over time(FIG. 10, Panel A), accompanied by a concomitant expansion of theresidual T cells (FIG. 10, Panel B) and the induction of T cellactivation markers (FIG. 10, Panel C). In DART-A treated samples, Tcells were expanded from around 7% to around 80% by 120 hours. T cellactivation measured by CD25 expression on CD4 and CD8 cells peaked at 72h and decreased by the 120 h timepoint.

Should it be necessary to replicate this example it will be appreciatedthat one of skill in the art may, within reasonable and acceptablelimits, vary the above-described protocol in a manner appropriate forreplicating the described results. Thus, the exemplified protocol is notintended to be adhered to in a precisely rigid manner.

Example 9 CD123 Surface Expression on Leukemic Blast Cells and StemCells in Primary Tissue Sample from ALL Patient

To define the CD123 expression pattern in ALL patient primary samples,cryopreserved primary ALL patient PBMC sample was evaluated for CD123surface expression on leukemic blast cells.

CD123 Expression in Leukemic Blast Cells in Peripheral BloodMononucleocytes (PBMC)

A total of 0.5×10⁶ peripheral blood mononucleocytes (PBMC)) from ahealthy donor and an ALL patient were evaluated for CD123 expression. Asshown in FIG. 11, Panels E-H, the vast majority of the cells from ALLbone marrow expressed CD123. Conversely, as expected in the normal donorB cells are CD123 negative and pDCs and monocytes are CD123 positive(FIG. 11, Panel D).

The T cell population was identified in the ALL patent sample bystaining the cells for CD4 and CD8. As shown in FIG. 12, Panel B, only asmall fraction of the total PBMCs in the ALL patient sample are T cells(approximately 0.5% are CD4 T cells and approximately 0.4% are CD8 Tcells.

Example 10 Autologous CTL Killing Assay Using ALL Patient PrimarySpecimens

Cryopreserved primary ALL specimen (peripheral blood mononucleocytes(PBMC)) were thawed in RPM 11640 with 10% FBS and allowed to recoverovernight at 37° C. in 5% CO₂. Cells were washed with assay medium (RPMI1640+10% FBS) and viable cell count was determined by Trypan Blueexclusion. 150,000 cells/well in 150 μL assay medium were added to96-well U-bottom plate (BD Biosciences). Sequence-optimized CD123×CD3bi-specific diabody (DART-A) was diluted to 10, 1 ng/mL and 50 μL ofeach dilution was added to each well (final volume=200 μL). A separateassay plate was set up for each time point (48, 72, 120 and 144 hours)and plates were incubated at 37° C. in a 5% CO₂ incubator. At each timepoint, cells were stained with CD4, CD8, CD25, CD45, CD33, and CD123antibodies. Labeled cells were analyzed in FACS Calibur flow cytometerequipped with CellQuest Pro acquisition software, Version 5.2.1 (BDBiosciences). Data analysis was performed using Flowjo v9.3.3 software(Treestar, Inc). T cell expansion was measured by gating on CD4+ andCD8+ populations and activation was determined by measuring CD25 MFI onthe CD4⁺ and CD8⁺-gated populations. Leukemic blast cell population wasidentified by CD45⁺CD33⁺ gating.

Autologous Tumor Cell Depletion, T Cell Expansion and Activation bySequence-Optimized CD123×CD3 Bi-Specific Diabody (DART-A) in PrimarySpecimens from ALL Patients

To determine the sequence-optimized CD123×CD3 bi-specific diabody(DART-A) mediated activity in ALL patient primary patient samples,patient samples were incubated with 1 ng/mL of DART-A and percentages ofleukemic blast cells and T cells were measured at different time pointsfollowing the treatment. Leukemic blast cells were identified byCD45⁺/CD33⁺ gating. Incubation of primary ALL bone marrow samples withDART-A resulted in depletion of the leukemic cell population over timecompared to untreated control or Control DART (FIG. 13, Panel H versusPanels F and G). When the T cells were counted (CD8 and CD4 staining)and activation (CD25 staining) were assayed, the T cells expanded andwere activated in the DART-A sample (FIG. 14, Panels I and L,respectively) compared to untreated or Control DART samples (FIG. 14,Panels H, G, K and J, respectively).

Example 11 CD123 Surface Expression on Leukemic Blast Cells and StemCells in Primary Tissue Sample from AML Patient 2

To define the CD123 expression pattern in AML patient 2 primary samples,cryopreserved primary AML patient bone marrow and PBMC samples wereevaluated for CD123 surface expression on leukemic blast cells.

CD123 Expression in Leukemic Blast Cells in Bone Marrow Mononucleocytes(BMNC)

A total of 0.5×10⁶ bone marrow mononucleocytes (BM MNC) and peripheralblood mononucleocytes (PBMC)) from an AML patient 2 were evaluated forleukemic blast cell identification. Leukemic blast cells were identifiedusing the myeloid markers CD33 and CD45. As shown in FIG. 15, Panel B,94% of the cells from AML bone marrow are leukemic blast cells. The Tcell population was identified by CD3 expression. As shown in FIG. 15,Panel C, approximately 15% of the cell from the AML bone marrow and PBMCsample are T cells.

Example 12 Autologous CTL Killing Assay Using AML Patient 2 PrimarySpecimens

Cryopreserved primary AML specimen (bone marrow mononucleocytes (BM MNC)and peripheral blood mononucleocytes (PBMC)) from AML patient 2 werethawed in RPMI 1640 with 10% FBS and allowed to recover overnight at 37°C. in 5% CO₂. Cells were washed with assay medium (RPMI 1640+10% FBS)and viable cell count was determined by Trypan Blue exclusion. 150,000cells/well in 150 μL assay medium were added to 96-well U-bottom plate(BD Biosciences). Sequence-optimized CD123×CD3 bi-specific diabody(DART-A) and control bi-specific diabody (Control DART) were diluted to0.1, and 0.01 ng/mL and 50 μL of each dilution was added to each well(final volume=2004). A separate assay plate was set up for each timepoint (48, 72, 120 and 144 hours) and plates were incubated at 37° C. ina 5% CO₂ incubator. At each time point, cells were stained with CD4,CD8, CD25, CD45, CD33, and CD123 antibodies. Labeled cells were analyzedin FACS Calibur flow cytometer equipped with CellQuest Pro acquisitionsoftware, Version 5.2.1 (BD Biosciences). Data analysis was performedusing Flowjo v9.3.3 software (Treestar, Inc). T cell expansion wasmeasured by gating on CD4+ and CD8+ populations and activation wasdetermined by measuring CD25 MFI on the CD4+ and CD8+-gated populations.Leukemic blast cell population was identified by CD45+CD33+ gating.

Autologous Tumor Cell Depletion, T Cell Expansion and Activation inPrimary Specimens from AML Patient 2

To determine the sequence-optimized CD123×CD3 bi-specific diabody(DART-A) mediated activity in AML patient primary patient 2 samples,patient samples were incubated with 0.1 or 0.01 ng/mL of DART-A andpercentages of leukemic blast cells and T cells were measured atdifferent time points following the treatment. Incubation of primary AMLbone marrow samples with DART-A resulted in depletion of the leukemiccell population over time (FIG. 16, Panel A), accompanied by aconcomitant expansion of the residual T cells (both CD4 and CD8) (FIG.16, Panel B and FIG. 16, Panel C, respectively). To determine if the Tcells were activated, cells were stained for CD25 or Ki-67, both markersof T cell activation. As shown in FIG. 17, Panels A and B, incubation ofprimary AML bone marrow samples with DART-A resulted in T cellactivation. These data represent the 144 h time point.

Intracellular Staining for Granzyme B and Perforin

To determine the intracellular levels of granzyme B and Perforin in Tcells, CTL assay was setup. After approximately 18 h, cells from theassay plate were stained with anti-CD4 and anti-CD8 antibodies byincubating for 30 minutes at 4° C. Following surface staining, cellswere incubated in 100 μl Fixation and Permeabilization buffer for 20 minat 4° C. Cells were washed with permeabilization/wash buffer andincubated in 50 μl of granzyme B and perforin antibody mixture preparedin 1× Perm/Wash buffer at 4 C for 30 minutes. Then cells were washedwith 250 μl Perm/Wash buffer and resuspended in Perm/Wash buffer forFACS acquisition.

Upregulation of Granzyme B and Perforin by Sequence-Optimized CD123×CD3Bi-Specific Diabody (DART-A) in T Cells During Redirected Killing.

To investigate the possible mechanism for sequence-optimized CD123×CD3bi-specific diabody (DART-A) mediated cytotoxicity by T cells,intracellular granzyme B and perforin levels were measured in T cellsafter the redirected killing. There was no upregulation of granzyme Band perforin when T cells were incubated with control bi-specificdiabody (Control DART). Upregulation of granzyme B and perforin levelsin both CD8 and CD4 T cells was observed with sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) (FIG. 17, Panels C and D).Interestingly, the upregulation was almost two-fold higher in CD8 Tcells compared to CD4 T cells (FIG. 17, Panel C and FIG. 17, Panel D).These data indicate that DART-A-mediated target cell killing wasmediated through granzyme B and perforin pathway.

Example 13 Sequence-Optimized CD123×CD3 Bi-Specific Diabody Cross-Reactswith Non-Human Primate CD123 and CD3 Proteins

In order to quantitate the extent of binding between sequence-optimizedCD123×CD3 bi-specific diabody (DART-A) and human or cynomolgus monkeyCD3, BIACORE™ analyses were conducted. BIACORE™ analyses measure thedissociation off-rate, kd. The binding affinity (KD) between an antibodyand its target is a function of the kinetic constants for association(on rate, ka) and dissociation (off-rate, kd) according to the formula:KD=[kd]/[ka]. The BIACORE™ analysis uses surface plasmon resonance todirectly measure these kinetic parameters. Recombinant human orcynomolgus monkey CD3 was directly immobilized to a support. Purifiedhuman or cynomolgus monkey CD123 was captured and immolbilized to asupport. The time course of dissociation was measured and a bivalent fitof the data conducted. Binding constants and affinity were obtainedusing a 1:1 binding fit. The results of the BIACORE™ analyses comparingbinding to human versus cynomologus monkey CD123 and CD3 proteins areshown in FIG. 18. Binding affinities to the cynomolgus monkey CD123(FIG. 18D) and CD3 (FIG. 18B) proteins is comparable to bindingaffinities for human CD123 (FIG. 18C) and CD3 (FIG. 18A) proteins.

Example 14 Autologus Monocyte Depletion In Vitro with Human andCynomolgus Monkey PBMCs

PBMCs from human or cynomolgus monkey whole blood samples were added toU-bottom plates at cell density of 200,000 cells/well in 150 μL of assaymedium. Dilutions of sequence-optimized CD123×CD3 bi-specific diabodies(DART-A or DART-A w/ABD) were prepared in assay medium. 50 μL of eachDART-A or DART-A w/ABD dilution was added to the plate containing PBMCsin duplicate wells. The plates were incubated for ˜18-24 h at 37° C.Supernatants were used to determine the cytotoxicity as described above.As shown in FIG. 19 (Panels A and B), depletion of pDCs cells wasobserved in both human (FIG. 19, Panel A) and cynomolgus monkey PBMCs(FIG. 19, Panel B). These results indicate that circulating pDC can beused as a pharmacodynamic marker for preclinical toxicology studies incynomolgus monkeys.

Should it be necessary to replicate this example it will be appreciatedthat one of skill in the art may, within reasonable and acceptablelimits, vary the above-described protocol in a manner appropriate forreplicating the described results. Thus, the exemplified protocol is notintended to be adhered to in a precisely rigid manner.

Example 15 Plasmacytoid Dendritic Cell Depletion in Cynomolgus MonkeysTreated with Sequence-Optimized CD123×CD3 Bi-Specific Diabody (DART-A)

As part of a dose-range finding toxicology study, cynomolgus monkeyswere administered sequence-optimized CD123×CD3 bi-specific diabody(DART-A) as 4-day infusions at doses of 0.1, 1, 10, 30 100, 300, or 1000ng/kg. The Control DART was administered at 100 ng/kg. To identify pDCsand monocyte populations in cynomolgus monkey PBMCs, cells were labeledwith CD14-FITC antibody. Monocytes were identified as the CD14⁺population and pDCs were identified as the CD14⁻CD123⁺ population. Asshown in FIG. 20 Panels K and L, the pDCs were depleted as early as 4days post infusion with as little as 10 ng/kg DART-A. No pDC depletionwas seen in the control bi-specific diabody-(Control DART) treatedmonkeys or the vehicle+carrier-treated monkeys at the 4 d time point(FIG. 20, Panels G, H, C and D, respectively). Cytokine levels ofinterferon-gamma, TNF-alpha, IL6, IL5, IL4 and IL2 were determined at 4hours after infusion. There was little to no elevation in cytokinelevels at the DART-A treated animals compared to Control DART orvehicle-treated animals.

FIG. 21 and FIG. 22 provide the results of the FACS analysis for B cells(CD20⁺) (FIG. 21, Panel A), monocytes (CD14⁺) (FIG. 21, Panel B), NKcells (CD159⁺CD16⁺) (FIG. 21, Panel C), pDC (CD123^(HI), CD14⁻) (FIG.21, Panel D), and T cells (total, CD4⁺, and CD8⁺) (FIG. 22, Panel A,FIG. 22, Panel B, and FIG. 22, Panel D, respectively).

Treatment of monkeys with Control DART had no noticeable effects on T orB lymphocytes, NK cells, monocytes and pDCs. Treatment of monkeys withDART-A at doses of 10 ng/kg/d or higher resulted in the abrogation ofpDCs (FIG. 21, Panel D). The depletion of pDC was complete and durable,returning to pre-dose levels several weeks after completion of dosing.Circulating levels of T lymphocytes decreased upon DART-Aadministration, but returned to pre-dose level by the end of each weeklycycle, suggesting changes in trafficking rather than true depletion.Both CD4 and CD8 T lymphocytes followed the same pattern. TheT-lymphocyte activation marker, CD69 (FIG. 22, Panel C), was onlymarginally positive among circulating cells and did not track withDART-A dosing. B lymphocytes, monocytes and NK cells fluctuated over thecourse of DART-A dosing with substantial variability observed amongmonkeys. A trend toward increased circulating levels of B lymphocytesand monocytes was observed in both monkeys at the highest doses.

In summary, the above results demonstrate the therapeutic efficacy ofthe sequence-optimized CD123×CD3 bi-specific diabody (DART-A). Thesequence-optimized CD123×CD3 bi-specific diabody (DART-A) may beemployed as a therapeutic agent for the treatment of multiple diseasesand conditions, including: AML, ABL (ALL), CLL, MDS, pDCL, mantel cellleukemia, hairy cell leukemia, Ricter transformation of CLL, Blasticcrisis of CML, BLL (subset are CD123+) (see Example 2); Autoimmune Lupus(SLE), allergy (basophils are CD123+), asthma, etc.

Example 16 Comparative Properties of Sequence-Optimized CD123×CD3Bi-Specific Diabody (DART-A) and Non-Sequence-Optimized CD123×CD3Bi-Specific Diabody (DART-B) Unexpected Advantage and Attributes of theSequence-Optimized CD123×CD3 Bi-Specific Diabodies

As discussed above, DART-A and DART-B are similarly designed and thefirst polypeptide of both constructs comprise, in the N-terminal toC-terminal direction, an N-terminus, a VL domain of a monoclonalantibody capable of binding to CD3 (VL_(CD3)), an intervening linkerpeptide (Linker 1), a VH domain of a monoclonal antibody capable ofbinding to CD123 (VH_(CD123)), a Linker 2, an E-coil Domain, and aC-terminus. Likewise, the second polypeptide of both constructscomprise, in the N-terminal to C-terminal direction, an N-terminus, a VLdomain of a monoclonal antibody capable of binding to CD123(VL_(CD123)), an intervening linker peptide (Linker 1), a VH domain of amonoclonal antibody capable of binding to CD3 (VH_(CD3)), a Linker 2, aK-coil Domain and a C-terminus.

As indicated in Example 1, both CD123×CD3 bi-specific diabodies werefound to be capable of simultaneously binding to CD3 and CD123.Additionally, as disclosed in Example 3 and in FIG. 4, Panels C and D,the two CD123×CD3 bi-specific diabodies exhibited a potent redirectedkilling ability with concentrations required to achieve 50% of maximalactivity (EC50s) in sub-ng/mL range, regardless of CD3 epitope bindingspecificity (DART-A versus DART-B) in target cell lines with high CD123expression. Thus, slight variations in the specific sequences of theCD123×CD3 bi-specific diabodies do not completely abrogate biologicalactivity.

However, in all cell lines tested, DART-A was found to be more activeand more potent at redirected killing than DART-B (see, e.g., FIG. 4,Panels A, C, and D). Thus DART-A exhibited an unexpected advantage oversimilar DART-B.

Example 17 Non-Human Primate Pharmacology of DART-A for the Treatment ofHematological Malignancies

The interleukin 3 (IL-3) receptor alpha chain, CD123, is overexpressedon malignant cells in a wide range of hematological malignancies (Munoz,L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is WidelyExpressed In Hematologic Malignancies,” Haematologica 86:1261-1269;Testa, U. et al. (2014) “CD123 Is A Membrane Biomarker And A TherapeuticTarget In Hematologic Malignancies,” Biomark. Res. 2:4) and isassociated with poor prognosis (Vergez, F. et al. (2011) “High Levels OfCD34+CD38low/−CD123+ Blasts Are Predictive Of An Adverse Outcome InAcute Myeloid Leukemia: A Groupe Ouest-Est Des Leucemies Aigues EtMaladies Du Sang (GOELAMS) Study,” Haematologica 96:1792-1798).Moreover, CD123 has been reported to be expressed by leukemia stem cells(LSC) (Jordan, C. T. et al. (2000) “The Interleukin-3 Receptor AlphaChain Is A Unique Marker For Human Acute Myelogenous Leukemia StemCells,” Leukemia 14:1777-1784; Jin, L. et al. (2009) “MonoclonalAntibody Mediated Targeting Of CD123, IL-3 Receptor Alpha Chain,Eliminates Human Acute Myeloid Leukemic Stem Cells,” Cell Stem Cell5:31-42), which is an attractive feature that enables targeting the rootcause of such diseases. Consistent with this conclusion, CD123 alsotakes part in an IL-3 autocrine loop that sustains leukemogenesis, asshown by the ability of a CD123-blocking monoclonal antibody to reduceleukemic stem cell engraftment and improve survival in a mouse model ofacute myelogenous leukemia (AML) (Jin, L. et al. (2009) “MonoclonalAntibody-Mediated Targeting Of CD123, IL-3 Receptor Alpha Chain,Eliminates Human Acute Myeloid Leukemic Stem Cells,” Cell Stem Cell5:31-42). In a phase 1 study in high-risk AML patients, however, themonoclonal antibody exhibited no anti-leukemic activity (Roberts, A. W.et al. (2010) “A Phase I Study Of Anti-CD123 Monoclonal Antibody (mAb)CSL360 Targeting Leukemia Stem Cells (LSC) In AML,” J. Clin. Oncol.28(Suppl):e13012). Thus, alternate CD123-targeting approaches, includingdepleting strategies are desired. Although CD123 is expressed by asubset of normal hematopoietic progenitor cells (HPC), hematopoieticstem cells (HSC) express little to no CD123 (Jordan, C. T. et al. (2000)“The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For HumanAcute Myelogenous Leukemia Stem Cells,” Leukemia 14:1777-1784; Jin, W.et al. (2009) “Regulation Of Th17 Cell Differentiation And EAE InductionBy MAP3K NIK,” Blood 113:6603-6610), indicating that CD123cell-depleting strategies allow reconstitution via normal hematopoiesis.

Enabling a patient's own T lymphocytes to target leukemic cellsrepresents a promising immunotherapeutic strategy for the treatment ofhematological malignancies. The therapeutic potential of this approachhas been attempted using blinatumomab (a bi-specific antibody-based BiTEhaving the ability to bond CD3 and the B cell CD19 antigen) in patientswith B cell lymphomas and B-precursor acute lymphoblastic leukemia(Klinger, M. et al. (2012) “Immunopharmacologic Response Of PatientsWith B-Lineage Acute Lymphoblastic Leukemia To Continuous Infusion Of TCell-Engaging CD19/CD3-Bispecific BiTE Antibody Blinatumomab,” Blood119:6226-6233; Topp, M. S. et al. (2012) “Long-Term Follow-Up OfHematologic Relapse-Free Survival In A Phase 2 Study Of Blinatumomab InPatients With MRD In B-Lineage ALL,” Blood 120:5185-5187; Topp, M. S. etal. (2011) “Targeted Therapy With The T-Cell-Engaging AntibodyBlinatumomab Of Chemotherapy-Refractory Minimal Residual Disease InB-Lineage Acute Lymphoblastic Leukemia Patients Results In High ResponseRate And Prolonged Leukemia-Free Survival,” J. Clin. Oncol.29:2493-2498).

The CD123×CD3 bi-specific diabody molecules of the present invention,such as DART-A, comprise an alternate bi-specific, antibody-basedmodality that offers improved stability and more robustmanufacturability properties (Johnson, S. et al. (2010) “Effector CellRecruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein LeadsTo Potent Tumor Cytolysis And In Vivo B-Cell Depletion,” J. Mol. Biol.399:436-449; Moore, P. A. et al. (2011) “Application Of Dual AffinityRetargeting Molecules To Achieve Optimal Redirected T-Cell Killing OfB-Cell Lymphoma,” Blood 117:4542-4551).

In order to demonstrate the superiority and effectiveness of theCD123×CD3 bi-specific diabody molecules of the present invention, thebiological activity of the above-described DART-A in in vitro andpreclinical models of leukemia was confirmed, and its pharmacokinetics,pharmacodynamics and safety pharmacology in cynomolgus macaques (Macacafascicularis) was assessed relative to either the above-describedControl DART (bi-specific for CD3 and fluorescein) or a “Control DART-2”that was bi-specific for CD123 and fluorescein).

Amino Acid Sequence of First Polypeptide Chainof “Control DART-2” (CD123VL - Linker - 4-4420VH - Linker - E-coil; linkers are underlined) (SEQ ID NO: 58):DFVMTQSPDS LAVSLGERVT MSCKSSQSLL NSGNQKNYLTWYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLTISSLQAEDVA VYYCQNDYSY PYTFGQGTKL EIK GGGSGGG GEVKLDETGG GLVQPGRPMK LSCVASGFTF SDYWMNWVRQSPEKGLEWVA QIRNKPYNYE TYYSDSVKGR FTISRDDSKSSVYLQMNNLR VEDMGIYYCT GSYYGMDYWG QGTSVTVSS G GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKAmino Acid Sequence of Second Polypeptide Chainof “Control DART-2” (4420VL - Linker - CD123VH - Linker - K-coil) (SEQ ID NO: 59):DVVMTQTPFS LPVSLGDQAS ISCRSSQSLV HSNGNTYLRWYLQKPGQSPK VLIYKVSNRF SGVPDRFSGS GSGTDFTLKISRVEAEDLGV YFCSQSTHVP WTFGGGTKLE IK GGGSGGGGEVQLVQSGAE LKKPGASVKV SCKASGYTFT DYYMKWVRQAPGQGLEWIGD IIPSNGATFY NQKFKGRVTI TVDKSTSTAYMELSSLRSED TAVYYCARSH LLRASWFAYW GQGTLVTVSS GGCGGGKVAA LKEKVAALKE KVAALKEKVA ALKE

Bifunctional ELISA

A MaxiSorp ELISA plate (Nunc) coated overnight with the soluble human orcynomolgus IL3R-alpha (0.5 μg/mL) in bicarbonate buffer was blocked with0.5% BSA; 0.1% Tween-20 in PBS (PBST/BSA) for 30 minutes at roomtemperature. DART-A molecules were applied, followed by the sequentialaddition of human CD3εδ-biotin and Streptavidin HRP (JacksonImmunoResearch). HRP activity was detected by conversion oftetramethylbenzidine (BioFX) as substrate for 5 min; the reaction wasterminated with 404/well of 1% H2504 and the absorbance read at 450 nm.

Surface Plasmon Resonance Analysis

The ability of DART-A to bind to human and cynomolgus monkey CD3 orCD123 proteins was analyzed using a BIAcore 3000 biosensor (GE,Healthcare) as described by Johnson, S. et al. (2010) (“Effector CellRecruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein LeadsTo Potent Tumor Cytolysis And In Vivo B-Cell Depletion,” J. Mol. Biol.399:436-449) and Moore, P. A. et al. (2011) (“Application Of DualAffinity Retargeting Molecules To Achieve Optimal Redirected T-CellKilling Of B-Cell Lymphoma,” Blood 117:4542-4551). Briefly, the carboxylgroups on the CMS sensor chip were activated with an injection of 0.2MN-ethyl-N-(3dietylamino-propyl) carbodiimide and 0.05MN-hydroxy-succinimide. Soluble CD3 or CD123 (1 μg/ml) was injected overthe activated CMS surface in 10 mM sodium-acetate, pH 5.0, at flow rate5 μL/min, followed by 1 M ethanolamine for deactivation. Bindingexperiments were performed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mMEDTA and 0.005% P20 surfactant. Regeneration of the immobilized receptorsurfaces was performed by pulse injection of 10 mM glycine, pH 1.5. KDvalues were determined by a global fit of binding curves to the Langmuir1:1 binding model (BIAevaluation software v4.1).

Cell Killing Assay

Cell lines used for cell killing assays were obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). PBMCs were isolated fromhealthy donor blood using the Ficoll-Paque Plus kit (GE Healthcare); Tcells were purified with a negative selection kit (Life Technologies).CD123 cell-surface density was determined using Quantum Simply Cellularbeads (Bangs Laboratories, Inc., Fishers, Ind.). Cytotoxicity assayswere performed as described by Moore, P. A. et al. (2011) (“ApplicationOf Dual Affinity Retargeting Molecules To Achieve Optimal RedirectedT-Cell Killing Of B-Cell Lymphoma,” Blood 117:4542-4551). Briefly,target cell lines (10⁵ cells/mL) were treated with serial dilutions ofDART-A or Control DART proteins in the presence of T cells at theindicated effector cells:target cells ratios and incubated at 37° C.overnight. Cell killing was determined as the release of lactatedehydrogenase (LDH, Promega) in culture supernatant. For flow-basedkilling, target cells were labeled with CMTMR (Life Technologies) andcell killing was monitored using a FACSCalibur flow cytometer. Data wereanalyzed by using PRISM® 5 software (GraphPad) and presented as percentcytotoxicity.

Cynomolgus Monkey Pharmacology

Non-human primate experiments were performed at Charles RiverLaboratories (Reno, Nev.), according to the guidelines of the localInstitutional Animal Care and Use Committee (IACUC). Purpose-bred, naïvecynomolgus monkeys (Macaca fascicularis) of Chinese origin (age range2.5-9 years, weight range of 2.7-5 kg) were provided with vehicle orDART-A via intravenous infusion through femoral and jugular ports usingbattery-powered programmable infusion pumps (CADD-Legacy®, SIMS Deltec,Inc., St. Paul, Minn.). Peripheral blood or bone marrow samples werecollected in anticoagulant containing tubes at the indicated timepoints. Cell-surface phenotype analyses were performed with an LSRFortessa analyzer (BD Biosciences) equipped with 488 nm, 640 nm and 405nm lasers and the following antibodies: CD4-V450, CD8-V450,CD123-PE-Cy7, CD45-PerCP, CD4-APC-H7, CD8-FITC, CD25-PE-Cy7, CD69-PerCP,PD-1-PE, TIM3-APC, CD3-Pacific Blue, CD95-APC, CD28-BV421, CD16-FITC,CD3-Alexa488, CD38-PE, CD123-PE-Cy7, CD117-PerCP-Cy5.5, CD34-APC,CD90-BV421, CD45RA-APC-H7 and CD33-APC (BD Biosciences). The absolutenumber of cells was determined using TruCOUNT (BD Biosciences). Serumlevels of IL-2, IL-4, IL-5, IL-6, TNF-α, and IFN-γ cytokines weremeasured with the Non-Human Primate Th1/Th2 Cytokine Cytometric BeadArray Kit (BD Bioscience). The concentration of DART-A in monkey serumsamples was measured using a sandwich immunoassay withelectrochemiluminescence detection (MesoScale Diagnostics, MSD,Rockville, Md.). Briefly, the assay plate (MSD) was coated withrecombinant human IL-3 Ra (R&D System) and blocked with 5% BSA.Calibration standards or diluted test samples were applied, followed bythe addition of a biotinylated monoclonal antibody exhibiting specificbinding for the above-described E-coil (SEQ ID NO:34) and K-coil (SEQ IDNO:35) domains of the molecule A SULFO-TAG™ labeled streptavidinconjugate (MSD) was added and the formation of complexes was analyzed inan MSD SECTOR® imager. DART-A concentrations were determined fromstandard curves generated by fitting light intensity data in afive-parameter logistic model.

Physicochemical characterization of the purified DART-A demonstrated ahomogeneous heterodimer with a molecular mass of 58.9 kDa (FIG. 23;FIGS. 24A-24B), which was stable at 2-8° C. for up to 12 months in PBS.SPR analysis demonstrated nearly identical binding affinities of DART-Ato the corresponding soluble human and cynomolgus monkey CD3 and CD123antigens (FIGS. 25A-25D and Table 7). Furthermore, DART-A simultaneouslybound both antigens in an ELISA format that employed human or monkeyCD123 for capture and human CD3 for detection (FIGS. 26A-26B), anddemonstrated similar binding to human and monkey T lymphocytes (FIGS.26C-26E). The data in Table 7 are averages of 3 independent experimentseach performed in duplicates.

TABLE 7 Equilibrium Dissociation Constants (K_(D)) for the Binding ofDART-A to Human and Cynomolgus Monkey CD3 and CD123 k_(a) (±SD) k_(d)(±SD) K_(D) (±SD) Antigens (M⁻¹s⁻¹) (s⁻¹) (nM) Human 5.7 (±0.6) × 10⁵5.0 (±0.9) × 10⁻³ 9.0 ± 2.3 CD3ε/δ Cynomolgus 5.5 (±0.5) × 10⁵ 5.0(±0.9) × 10⁻³ 9.2 ± 2.3 CD3ε/δ Human 1.6 (±0.4) × 10⁶ 1.9 (±0.4) × 10⁻⁴0.13 ± 0.01 CD123-His Cynomolgus 1.5 (±0.3) × 10⁶ 4.0 (±0.7) × 10⁻⁴ 0.27± 0.02 CD123-His

DART-A Mediates Redirected Killing by Human or Cynomolgus Monkey TLymphocytes

DART-A mediated redirected target cell killing by human or monkeyeffector cells against CD123+ Kasumi-3 leukemic cell lines (FIG.27A-27D), which was accompanied by induction of activation markers. Noactivity was observed against CD123-negative targets (U937 cells) orwith Control DART, indicating that T cell activation is strictlydependent upon target cell engagement and that monovalent engagement ofCD3 by DART-A was insufficient to trigger T cell activation. Since CD123is expressed by subsets of normal circulating leukocytes, including pDCsand monocytes (FIG. 27E), the effect of DART-A were further investigatedin normal human and monkey's PBMCs.

A graded effect was observed among human PBMC, with a dose-dependentrapid depletion of CD14⁻CD123^(high) cells (pDC and basophils) observedas early as 3 hours following initiation of treatment, while monocytes(CD14+ cells) remained unaffected at this time point (FIGS. 27F-27G).CD14⁻CD123^(high) cells depletion increased over time across all DART-Amolecule concentrations, while monocytes were slightly decreased by 6hours and depleted only after 18 hours and at the concentrations higherthan 1 ng/mL. Incubation of monkey PBMCs with DART-A resulted in acomparable dose-dependent depletion of CD14⁻CD123^(high) cells (FIG.27H), further supporting the relevance of this species for DART-Apharmacology (CD14+ monkey cells express little to no CD123 and were notdepleted).

Pharmacokinetics of DART-A in Cynomolgus Monkeys

The cynomolgus monkey was selected as an appropriate pharmacologicalmodel for DART-A analysis based on the equivalent distribution of bothtarget antigens in this species compared to humans based onimmunohistochemistry with the precursor mAbs, consistent with publishedinformation (Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain(CD123) Is Widely Expressed In Hematologic Malignancies,” Haematologica86:1261-1269; Korpelainen, E. I. et al. (1996) “IL-3 ReceptorExpression, Regulation And Function In Cells Of The Vasculature,”Immunol. Cell Biol. 74:1-7).

The study conducted in accordance with the present invention included 6treatment groups consisting of 8 cynomolgus monkeys per group (4 males,4 females) (Table 8). All groups received vehicle control for the firstinfusion; then vehicle or DART-A were administered intravenously for 4weekly cycles. Group 1 animals received vehicle control for all 4subsequent infusions, whereas Groups 2-5 received weekly escalatingdoses of DART-A for 4 days a week for all subsequent infusions. Group 6animals were treated with 7-day uninterrupted weekly escalating doses ofDART-A for all infusions. The 4-day-on/3-day-off and 7-day-on scheduleswere designed to distinguish between durable from transient effectsassociated with DART-A administration. Two males and 2 females per groupwere sacrificed at the end of the treatment phase (Day 36), while theremaining monkeys were necropsied after a 4-week recovery (Day 65). Asubset of monkeys developed anti-drug antibodies (ADA) directed againstthe humanized Fv of both CD3 and CD123 and the data points following theappearance of ADA were excluded from the PK analysis. All monkeys wereexposed to DART-A during the study period.

TABLE 8 DART-A Infusion (4-day-on/3-day-off) (7-day-on) ng/kg/dayng/kg/day Vehicle [ng/kg/4 days] [ng/kg/7days] Infusion Study GroupGroup Group Group Group Group No. Days 1 2 3 4 5 6 1 1 Vehicle VehicleVehicle Vehicle Vehicle Vehicle 2 8 Vehicle 100 100 100 100 100 [400][400] [400] [400] [700] 3 15 Vehicle 100 300 300 300 300 [400] [1200][1200] [1200] [2100] 4 22 Vehicle 100 300 600 600 600 [400] [1200][2400] [2400] [4200] 5 29 Vehicle 100 300 600 1000 1000 [400] [1200][2400] [4000] [7000] Recovery 36-65

A two-compartment model was used to estimate PK parameters (Table 9 andFIG. 28). T_(1/2)α was short (4-5 min), reflecting rapid binding tocirculating targets; T_(1/2)β was also rapid, as expected for a moleculeof this size, which is subject to renal clearance. Analysis of serumsamples collected at the end of each infusion from group 6 monkeysshowed a dose-dependent increase in DART-A C_(max). In Table 9, Vehiclewas PBS, pH 6.0, containing 0.1 mg/mL recombinant human albumin, 0.1mg/mL PS-80, and 0.24% benzyl alcohol was used for all vehicle infusionsduring the first 4 days of each infusion week followed the sameformulation without benzyl alcohol for the remaining 3 days of eachweekly infusion. DART-A was administered for the indicated times as acontinuous IV infusion of a solution of PBS, pH 6.0, containing 0.1mg/mL recombinant human albumin, 0.1 mg/mL PS-80, and 0.24% benzylalcohol at the required concentration.

TABLE 9 Two-Compartment Analysis of PK Parameters of DART-A inCynomolgus Monkeys 300 ng/kg/d 600 ng/kg/d Attribute (mean ± SD) (mean ±SD) C_(max) (pg/mL) 77.4 ± 9.4  113.8 ± 33.5  AUC (h*pg/mL) 7465 ± 913 11188 ± 3282  V_(ss) (L/kg) 1.078 ± 0.511 2.098 ± 1.846 t_(1/2), alpha(h)  0.07 ± 0.018 0.067 ± 0.023 t½, beta (h) 13.79 ± 4.928 21.828 ±18.779 MRT (h)  6.73 ± 3.327 9.604 ± 8.891

Cytokine Release in DART-A-Treated Cynomolgus Monkeys

Given the T cell activation properties of DART-A, an increase incirculating cytokines accompanying the infusion was anticipated and alow starting dose was therefore used as a “desensitization” strategy,based on previous experience with similar compounds (see, e.g., Topp, M.S. et al. (2011) “Targeted Therapy With The T-Cell-Engaging AntibodyBlinatumomab Of Chemotherapy-Refractory Minimal Residual Disease InB-Lineage Acute Lymphoblastic Leukemia Patients Results In High ResponseRate And Prolonged Leukemia-Free Survival,” J. Clin. Oncol.29:2493-2498; Bargou, R. et al. (2008) “Tumor Regression In CancerPatients By Very Low Doses Of A T Cell-Engaging Antibody,” Science321:974-977). Of the cytokine tested, IL-6 demonstrated the largestchanges upon infusion, albeit transient in nature, of minimal magnitudeand with large inter-animal and inter-group variations (FIGS. 29A-29C).Small, transient increases in IL-6 were also observed after vehicleinfusions (all Group 1 and all Day 1 infusions), indicating asensitivity of this cytokine to manipulative stress. Nonetheless,DART-A-dependent increases (<80 μg/mL) in serum IL-6 were seen in somemonkeys following the first DART-A infusion (100 ng/kg/day), whichreturned to baseline by 72 hours. Interestingly, the magnitude of IL-6release decreased with each successive DART-A infusion, even when thedose level was increased to up to 1000 ng/kg/day. Minimal and transientDART-A-related increases in serum TNF-α (<10 μg/mL) were also observed;as with IL-6, the largest magnitude in TNF-α release was observedfollowing the first infusion. There were no DART-A-related changes inthe levels of IL-5, IL-4, IL-2, or IFN-γ throughout the study whencompared with controls. In conclusion, cytokine release in response totreatment of monkeys with DART-A was minimal, transient and representeda first-dose effect manageable via intra-subject dose escalation.

DART-A-Mediated Depletion of Circulating CD14⁻/CD123⁺ Leukocytes In Vivo

The circulating absolute levels of CD14-/CD123+ cells were measuredthroughout the study as a pharmacodynamic endpoint. While the number ofCD123⁺ cells in control Group 1 remained stable over time, DART-Atreatment was associated with extensive depletion of circulatingCD14-/CD123+ cells (94-100% from prestudy baseline) observable from thefirst time point measured (72 hours) following the start of the firstDART-A infusion (100 ng/kg/day) in all animals across all activetreatment groups (FIGS. 30A-30C). The depletion was durable, as itpersisted during the 3-day weekly dosing holiday in Group 2-5, returningto baseline levels only during the prolonged recovery period. Toeliminate the possibility of DART-A masking or modulating CD123 (anunlikely scenario, given the low circulating DART-A levels), pDCs wereenumerated by the orthogonal marker, CD303. Consistent with the CD123data, CD303+ pDC were similarly depleted in monkeys treated with DART-A(FIGS. 30D-30F).

Circulating T-Lymphocyte Levels, Activation and Subset Analysis

In contrast to the persistent depletion of circulating CD123+ cells,DART-A administered on the 4-day-on/3-day-off schedule (Groups 2-5) wereassociated with weekly fluctuations in circulating T cells, whileadministration as continuous 7-day infusions resulted in similarlydecreased circulating T cell levels following the first administrationthat slowly recovered without fluctuation even during the dosing period(FIGS. 31A-31C). The difference between the two dosing strategiesindicates that the effect of DART-A on T lymphocytes is consistent withtrafficking and/or margination, rather than depletion. Followingcessation of dosing, T cells rebounded to levels approximately 2-foldhigher than baseline for the duration of the recovery period. Infusionof DART-A was associated with an exposure-dependent, progressiveincreased frequency of T cells expressing the late activation marker,PD-1, particularly in CD4+ cells, with dose Group 6 displaying thehighest overall levels (FIGS. 31D-31I and FIGS. 32A-32F and FIGS.33A-33F). Tim-3, a marker associated with T cell exhaustion, was notdetected on CD4+ T cells and only at low frequency among CD8+ cells(5.5-9.7%) and comprising 20.5-35.5% of the CD8+/PD-1+ double-positivecells. There was no consistent change in the early T cell activationmarker, CD69, and only modest variations in CD25 expression amongcirculating cells.

To rule out exhaustion after in vivo exposure, the ex vivo cytotoxicpotential of effector cells isolated from cynomolgus monkeys receivingmultiple infusions of DART-A was compared to that of cells from naïvemonkeys. As shown in FIG. 34, PBMC isolated from DART-A-treated monkeysshow cytotoxicity comparable to that of cells isolated from naïvemonkeys, indicating that in vivo exposure to DART-A does not negativelyimpact the ability of T cells to kill target cells.

DART-A exposure increased the relative frequency of central memory CD4cells and effector memory CD8+ cells at the expense of the correspondingnaïve T cell population (FIGS. 35A-35F and FIGS. 32A-32F and FIGS.33A-33F), indicating that DART-A exposure promoted expansion and/ormobilization of these cells.

Effects on Hematopoiesis and Bone Marrow Precursors

DART-A was well tolerated in monkeys at all doses tested; however,reversible reductions in red cell parameters were observed at thehighest doses (FIGS. 36A-36C). Frequent blood sampling could have been apotential contributing factor, since vehicle-treated animals showed amodest decline in red cell mass. Reticulocyte response was observed inall animals; at the highest exposure (Group 6), however, the responseappeared slightly less robust for similar decrease in red cell mass(FIGS. 36D-36F). Morphological analysis of bone marrow smears throughoutthe study was unremarkable. Flow cytometry analysis, however, revealedthat the frequency of CD123+ cells within the immature lineage-negative(Lin−) bone marrow populations decreased in DART-A-treated animals atthe end of the dosing period, returning to baseline levels by the end ofthe recovery time (FIG. 37A-37B). HSC (defined asLin−/CD34+/CD38−/CD45RA−/CD90+ cells (Pang, W. W. et al. (2011) “HumanBone Marrow Hematopoietic Stem Cells Are Increased In Frequency AndMyeloid-Biased With Age,” Proc. Natl. Acad. Sci. (U.S.A.)108:20012-20017)) showed large inter-group variability; Group 4-6DART-A-treated monkeys show some apparent reduction compared to thecorresponding pre-dose levels, however, no decrease was seen in alltreated groups compared to vehicle-treated animals. These data indicatethat HSC are less susceptible to targeting by DART-A and are consistentwith the observed reversibility of the negative effects of DART-Atreatment on hematopoiesis.

As demonstrated above, with respect to infusions for 4 weeks on a4-day-on/3-day-off weekly schedule or a 7-day-on schedule at startingdoses of 100 ng/kg/day that were escalated stepwise weekly to 300, 600,and 1,000 ng/kg/day, the administration of DART-A to cynomolgus monkeyswas well tolerated. Depletion of circulating CD123+ cells, includingpDCs, was observed after the start of the first administration andpersisted throughout the study at all doses and schedules. Reversiblereduction in bone marrow CD123+ precursor was also observed. Cytokinerelease, as significant safety concern with CD3-targeted therapies,appeared manageable and consistent with a first-dose effect. Modestreversible anemia was noted at the highest doses, but no other (on- oroff-target) adverse events were noted.

The cynomolgus monkey is an appropriate animal model for thepharmacological assessment of DART-A, given the high homology betweenthe orthologs and the ability of DART-A to bind with similar affinity tothe antigens and mediate redirected T cell killing in both species.Furthermore, both antigens are concordantly expressed in monkeys andhumans, including similar expression by hematopoietic precursors and inthe cytoplasm of the endothelium of multiple tissues. Minor exceptionsare the expression in testicular Leydig cells in humans but not monkeysand low-to-absent CD123 in monkey monocytes compared to humans.

A primary concern associated with therapeutic strategies that involve Tcell activation includes the release of cytokines and off-targetcytotoxic effects. A recent study with a CD3×CD123 bi-specific scFvimmunofusion construct with bivalent CD3 recognition demonstratedanti-leukemic activity in vitro, but caused non-specific activation of Tcells and IFN-γ secretion (Kuo, S. R. et al. (2012) “Engineering ACD123×CD3 Bispecific scFv Immunofusion For The Treatment Of Leukemia AndElimination Of Leukemia Stem Cells,” Protein Eng. Des. Sel. 25:561-569).The monovalent nature of each binding arms and the highly homogeneousmonomeric form of DART-A ensure that T cell activation dependsexclusively upon target cell engagement: no T cell activation wasobserved in the absence of target cells or by using a Control DARTmolecule that included only the CD3-targeting arm. Furthermore, highdoses (up to 100 ug/kg/day) of the Control DART molecule did not triggercytokine release in cynomolgus monkeys.

The DART-A molecule starting dose of 100 ng/kg/day was well tolerated,with minimal cytokine release. Cytokine storm, however, did occur with ahigh starting dose (5 μg/kg/day); however, such dose could be reachedsafely via stepwise weekly dose escalations, indicating thatDART-A-mediated cytokine release appears to be primarily a first-doseeffect. Depletion of the CD123+ target cells, thereby eliminating asource of CD3 ligation, may explain the first-dose effect: nearlycomplete CD123+ cell depletion was observed at doses as low as 3-10ng/kg/day, indicating that cytokine release in vivo follows a shifteddose-response compared to cytotoxicity. Dose-response profiles forcytotoxicity and cytokine release by human T cells were also consistentwith this observation.

T cell desensitization, in which DART-A-induced PD1 upregulation mayplay a role, appears to also contribute to limit cytokine release afterthe first infusion of DART-A. Recent studies show that increased PD-1expression after antigen-induced arrest of T cells at inflammation sitescontributes, through interactions with PD-L1, to terminating the stopsignal, thus releasing and desensitizing the cells (Honda, T. et al.(2014) “Tuning Of Antigen Sensitivity By T Cell Receptor-DependentNegative Feedback Controls T Cell Effector Function In InflamedTissues,” Immunity 40:235-247; Wei, F. et al. (2013) “Strength Of PD-1Signaling Differentially Affects T-Cell Effector Functions,” Proc. Natl.Acad. Sci. (U.S.A.) 110:E2480-E2489). The PD-1 countering of TCRsignaling strength is not uniform: while proliferation and cytokineproduction appear most sensitive to PD-1 inhibition, cytotoxicity is theleast affected (Wei, F. et al. (2013) “Strength Of PD-1 SignalingDifferentially Affects T-Cell Effector Functions,” Proc. Natl. Acad.Sci. (U.S.A.) 110:E2480-E2489). Consistently, the ex vivo cytotoxicpotential of T cells from monkeys exposed to multiple infusions ofDART-A was comparable to that of T cells from naïve monkeys, despiteincreased PD-1 levels in the former. Furthermore, PD-1 upregulation wasnot accompanied by TIM3 expression, a hallmark of T cell exhaustion, asshown for T cells exposed to protracted stimulation with CD3 antibodiesor chronic infections (Gebel, H. M. et al. (1989) “T Cells From PatientsSuccessfully Treated With OKT3 Do Not React With The T-Cell ReceptorAntibody,” Hum. Immunol. 26:123-129; Wherry, E. J. (2011) “T CellExhaustion,” Nat. Immunol. 12:492-499).

The depletion of circulating CD123+ cells in DART-A-treated monkeys wasrapid and persisted during the weekly dosing holidays in the4-day-on/3-day-off schedule, consistent with target cell elimination. Incontrast, the transient fluctuations in circulating T cells were likelythe result of trafficking from/to tissues and lymphoid organs as afunction of DART-A. DART-A exposure promotes the expansion and/ormobilization of antigen experienced T lymphocytes, cells thatpreferentially home to tissues and more readily exert cytotoxic effectorfunction (Mirenda, V. et al. (2007) “Physiologic And Aberrant RegulationOf Memory T-Cell Trafficking By The Costimulatory Molecule CD28,” Blood109:2968-2977; Marelli-Berg, F. M. et al. (2010) “Memory T-CellTrafficking: New Directions For Busy Commuters,” Immunology130:158-165).

Depletion of CD123+ normal cells may carry potential liabilities. pDCsand basophils express high levels of CD123, compared to lower levels inmonocytes and eosinophils (Lopez, A. F. et al. (1989) “ReciprocalInhibition Of Binding Between Interleukin 3 And Granulocyte MacrophageColony-Stimulating Factor To Human Eosinophils,” Proc. Natl. Acad. Sci.(U.S.A.) 86:7022-7026; Munoz, L. et al. (2001) “Interleukin-3 ReceptorAlpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies,”Haematologica 86:1261-1269; Masten, B. J. et al. (2006)“Characterization Of Myeloid And Plasmacytoid Dendritic Cells In HumanLung,” J. Immunol. 177:7784-7793; Korpelainen, E. I. et al. (1995)“Interferon-Gamma Upregulates Interleukin-3 (IL-3) Receptor ExpressionIn Human Endothelial Cells And Synergizes With IL-3 In Stimulating MajorHistocompatibility Complex Class II Expression And Cytokine Production,”Blood 86:176-182). pDCs have been shown to play a role in the control ofcertain viruses in mouse or monkey models of infection, although they donot appear critical for controlling the immune response to flu (Colonna,M. et al. (1997) “Specificity And Function Of Immunoglobulin SuperfamilyNK Cell Inhibitory And Stimulatory Receptors,” Immunol. Rev.155:127-133; Smit, J. J. et al. (2006) “Plasmacytoid Dendritic CellsInhibit Pulmonary Immunopathology And Promote Clearance Of RespiratorySyncytial Virus,” J. Exp. Med. 203:1153-1159). In tumor models, pDCs maypromote tumor growth and metastasis, while pDC depletion resulted intumor inhibition (Sawant, A. et al. (2012) “Depletion Of PlasmacytoidDendritic Cells Inhibits Tumor Growth And Prevents Bone Metastasis OfBreast Cancer Cells,” J. Immunol. 189:4258-4265). Transient, modest,dose-independent facial swelling was observed in some monkeys treatedwith DART-A; however, no increased histamine levels were observed inthese monkeys or when human basophils were lysed via DART-A-mediated Tcell killing. Monocyte depletion may carry increased risks of infection;the consequence of pDC, basophil or eosinophils depletion in humansshould thus be monitored.

Committed hematopoietic precursors that express CD123, such as thecommon myeloid precursor (CMP) (Jordan, C. T. et al. (2000) “TheInterleukin-3 Receptor Alpha Chain Is A Unique Marker For Human AcuteMyelogenous Leukemia Stem Cells,” Leukemia 14:1777-1784; Rieger, M. A.et al. (2012) “Hematopoiesis,” Cold Spring Harb. Perspect. Biol.4:a008250), may be targeted by DART-A, a possible explanation for themodest anemia observed following administration of DART-A at the highestdose. The erythropoietic reticulocyte response appeared to function atall DART-A dose levels; however, for commensurate drops in red cellparameters, animals subjected to the greatest DART-A exposure (Group 6,7-day-on infusion) showed a reduced reticulocyte response, suggesting apossible cytotoxic activity on precursors (e.g., CMP). The effect wasreversible following cessation of DART-A treatment, consistent withrepopulation from spared CD123low/negative HSC.

Alternate approaches for depletion of CD123+ cells include asecond-generation CD123-specific Fc-enhanced monoclonal antibody (Jin,L. et al. (2009) “Monoclonal Antibody-Mediated Targeting Of CD123, IL-3Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic StemCells,” Cell Stem Cell 5:31-42; Roberts, A. W. et al. (2010) “A Phase IStudy Of Anti-CD123 Monoclonal Antibody (mAb) CSL360 Targeting LeukemiaStem Cells (LSC) In AML,” J. Clin. Oncol. 28(Suppl):e13012), IL-3 bounddiphtheria toxin (Frankel, A. et al. (2008) “Phase I Clinical Study OfDiphtheria Toxin-Interleukin 3 Fusion Protein In Patients With AcuteMyeloid Leukemia And Myelodysplasia,” Leuk. Lymphoma 49:543-553),cytokine-induced killer (CIK) cells expressing CD123-specific chimericantigen receptors (CAR) (Tettamanti, S. et al. (2013) “Targeting OfAcute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected WithA Novel CD123-Specific Chimeric Antigen Receptor,” Br. J. Haematol.161:389-401) and CD123 CAR T cells (Gill, S. et al. (2014) “EfficacyAgainst Human Acute Myeloid Leukemia And Myeloablation Of NormalHematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-ModifiedT Cells,” Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “T CellsExpressing CD123-Specific Chimeric Antigen Receptors Exhibit SpecificCytolytic Effector Functions And Antitumor Effects Against Human AcuteMyeloid Leukemia,” Blood 122:3138-3148). CAR T cells exhibited potentleukemic blast cell killing in vitro and anti-leukemic activity in axenogeneic model of disseminated AML (Mardiros, A. et al. (2013) “TCells Expressing CD123-Specific Chimeric Antigen Receptors ExhibitSpecific Cytolytic Effector Functions And Antitumor Effects AgainstHuman Acute Myeloid Leukemia,” Blood 122:3138-3148). A recent studyreported ablation of normal hematopoiesis in NSG mice engrafted withhuman CD34+ cells following CD123 CAR T cell transfer (Gill, S. et al.(2014) “Efficacy Against Human Acute Myeloid Leukemia And MyeloablationOf Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen ReceptorModified T Cells,” Blood 123(15): 2343-2354), although others have notobserved similar effects in vitro or in vivo (Tettamanti, S. et al.(2013) “Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced KillerCells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor,”Br. J. Haematol. 161:389-401; Pizzitola, I. et al. (2014) “ChimericAntigen Receptors Against CD33/CD123 Antigens Efficiently Target PrimaryAcute Myeloid Leukemia Cells in vivo,” Leukemiadoi:10.1038/leu.2014.62). In the above-discussed experiments, depletionof CD123+ bone marrow precursor populations was observed, but reversedduring recovery; furthermore, depletion of this minority populationresulted in no changes in bone marrow cellularity or erythroid tomyeloid cell (E:M) ratio at all DART-A dose levels tested. Thesedifferences underscore the potential advantages of DART-A over celltherapies, as it provides a titratable system that relies on autologousT cells in contrast to “supercharged” ex vivo transduced cells that maybe more difficult to control. CD123 is overexpressed in severalhematologic malignancies, including AML, hairy cell leukemia, blasticplasmacytoid dendritic cell neoplasms (BPDCNs), a subset of B-precursoracute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia,Hodgkin's disease Reed-Stemberg cells, as well as in myelodysplasticsyndrome and systemic mastocytosis (Kharfan-Dabaja, M. A. et al. (2013)“Diagnostic And Therapeutic Advances In Blastic Plasmacytoid DendriticCell Neoplasm: A Focus On Hematopoietic Cell Transplantation,” Biol.Blood Marrow Transplant. 19:1006-1012; Florian, S. et al. (2006)“Detection Of Molecular Targets On The Surface Of CD34+/CD38− Stem CellsIn Various Myeloid Malignancies,” Leuk. Lymphoma 47:207-222; Munoz, L.et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is WidelyExpressed In Hematologic Malignancies,” Haematologica 86:1261-1269;Fromm, J. R. (2011) “Flow Cytometric Analysis Of CD123 Is Useful ForImmunophenotyping Classical Hodgkin Lymphoma,” Cytometry B Clin. Cytom.80:91-99). The predictable pharmacodynamic activity and manageablesafety profile observed in non-human primates further supports theclinical utility and efficacy of DART-A as immunotherapy for thesedisorders.

In sum, DART-A is an antibody-based molecule engaging the CD3E subunitof the TCR to redirect T lymphocytes against cells expressing CD123, anantigen up-regulated in several hematological malignancies. DART-A bindsto both human and cynomolgus monkey's antigens with similar affinitiesand redirects T cells from both species to kill CD123+ cells. Monkeysinfused 4 or 7 days a week with weekly escalating doses of DART-A showeddepletion of circulating CD123+ cells 72 h after treatment initiationthat persisted throughout the 4 weeks of treatment, irrespective ofdosing schedules. A decrease in circulating T cells also occurred, butrecovered to baseline before the subsequent infusion in monkeys on the4-day dose schedule, consistent with DART-A-mediated mobilization.DART-A administration increased circulating PD1+, but not TIM-3+, Tcells; furthermore, ex vivo analysis of T cells from treated monkeysexhibited unaltered redirected target cell lysis, indicating noexhaustion. Toxicity was limited to a minimal transient release ofcytokines following the DART-A first infusion, but not after subsequentadministrations even when the dose was escalated, and a minimalreversible decrease in red cell mass with concomitant reduction inCD123+ bone marrow progenitors. Clinical testing of DART-A inhematological malignancies appears warranted.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

1-20. (canceled)
 21. A method of treating a disease or conditionassociated with or characterized by the expression of CD123, whereinsaid disease or condition is cancer or an inflammatory condition,comprising administering to a subject in need thereof a bi-specificdiabody capable of specific binding to an epitope of CD123 and to anepitope of CD3, wherein: I. said bi-specific diabody comprises a firstpolypeptide chain and a second polypeptide chain, covalently bonded toone another, and wherein: (A) said first polypeptide chain comprises theamino acid sequence of SEQ ID NO:1; and (B) said second polypeptidechain comprises the amino acid sequence of SEQ ID NO:3; and II. saidbi-specific diabody is administered at a dose of between about 0.3 ng/kgper day and about 1,000 ng/kg per day.
 22. The method of claim 21,wherein the administered dose escalates over the first quarter of thetreatment regimen.
 23. The method of claim 21, wherein said bi-specificdiabody is administered at a dose of between about 30 ng/kg per day andabout 90 ng/kg per day.
 24. The method of claim 21, wherein saidbi-specific diabody is administered at a dose of between about 100 ng/kgper day and about 300 ng/kg per day.
 25. The method of claim 21, whereinsaid bi-specific diabody is administered at a dose of between about 200ng/kg per day and about 600 ng/kg per day.
 26. The method of claim 21,wherein said bi-specific diabody is administered at a dose of 100 ng/kgper day.
 27. The method of claim 21, wherein said bi-specific diabody isadministered at a dose of 300 ng/kg per day.
 28. The method of claim 21,wherein said bi-specific diabody is administered at a dose of 500 ng/kgper day.
 29. The method of claim 21, wherein said bi-specific diabody isadministered on day 1, day 2, day 3, and day 4 of a given week, and isnot administered on day 5, day 6 and day 7 of the given week.
 30. Themethod of claim 21, wherein said disease or condition is cancer, andwherein said cancer is selected from the group consisting of: acutemyeloid leukemia (AML), chronic myelogenous leukemia (CML), blasticcrisis of CML, Abelson oncogene associated with CML (Bcr-ABLtranslocation), myelodysplasia syndrome (MDS), acute B lymphoblasticleukemia (B-ALL), chronic lymphocytic leukemia (CLL), Richter'ssyndrome, Richter's transformation of CLL, hairy cell leukemia (HCL),blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin'slymphoma (NHL), mantel cell leukemia (MCL), small lymphocytic lymphoma(SLL), Hodgkin lymphoma, systemic mastocytosis, and Burkitt's lymphoma.31. The method of claim 30, wherein said cancer is AML.
 32. The methodof claim 21, wherein said disease or condition is an inflammatorycondition, and wherein said inflammatory condition is selected from thegroup consisting of: Autoimmune Lupus (SLE), allergy, asthma, andrheumatoid arthritis.